Diagnostic method

ABSTRACT

The disclosure is directed to a method of diagnosing a prostate condition in a subject by determining, in a sample obtained from a subject, levels of a plurality of constituents selected from the group consisting of Ca, K, Mg, Zn, Ag, AI, Au, B, Ba, Bi, Br, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, La, Li, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, Tl, U, Y, and Zr. A combination of the levels of the plurality of constituents in the sample is compared with a combination of control levels of the same plurality of constituents. A difference between the combinations is indicative of the prostate condition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/312,422, filed Nov. 18, 2016, the entire contents of which areincorporated herein by reference

FIELD OF THE INVENTION

The present invention relates to a sensitive diagnostic method forestablishing a prostate condition.

BACKGROUND OF THE INVENTION Description of the Related Art

Prostate cancer (PCa) remains the second most common cancer worldwidefor males with an estimated 900,000 new cases diagnosed in 2008 (FerlayJ, et al. Estimates of cancer incidence and mortality in Europe in 2008,European Journal of Cancer, 2010 46:765-781). According to the CancerResearch UK, PCa is the most common cancer in males in the UK,accounting for 41,000 of new cases of cancer in males every year. In2008-2010 25% of PCa cases in the UK are diagnosed in men under the ageof 65 (CancerStats, Incidence 2009-UK, CRUK May 2012).

Prostate cancer normally causes no symptoms until the cancer has grownlarge enough to put pressure on the urethra. Symptoms can include weakurinal flow, frequent urination, pain when passing urine etc. Due to thefact that benign prostate conditions such as inflammation, infection andbenign prostatic hyperplasia are common in men over the age of 50 andproduce similar symptoms, discrimination between prostate cancer andbenign prostatic conditions presents a challenge to current diagnosticmethods. Currently, there is no single, effective screening test toaccurately diagnose prostate cancer in men. The most commonly used PCadiagnostic methods today include the serum prostate-specific antigenanalysis (PSA), the digital rectal examination (DRA), and theultrasound-guided prostate biopsy sampling (Horwich A, et al. Prostatecancer: ESMO Clinical Practice Guidelines for diagnosis, treatment andfollow-up. Annals of Oncology 21 (Supplement 5): v129-v133, 2010).Despite the years of research the specificity and sensitivity of the PSAbased multi-step diagnostic approach is still highly inaccurate. Forexample, in the European Randomized Study 75.9% of men who underwent abiopsy because of an elevated PSA value had no cancer (Schroeder F, etal. Screening and Prostate-Cancer Mortality in a Randomized EuropeanStudy, N Engl J Med 2009; 360:1320). In addition, a needle biopsy is aninvasive and painful procedure with side effects such as prostatitis andblood in urine or semen. Also, many men find the DRA and the needlebiopsy embarrassing. In addition to high level of false-positiveresults, smaller tumors can be missed by current methods with fatalconsequences because prostate tumors have the potential to suddenly growand metastasize.

There is constant search for novel biomarkers to improve specificity ofPCa detection. For example, one of such biomarkers currently underclinical investigations is the prostate specific non-coding mRNA marker,PCA3, measured in urine sediment obtained after prostatic massage(Heidenreich A, et al. Guidelines on Prostate Cancer, EuropeanAssociation of Urology 2010). So far, however, none of theinvestigational biomarkers are being used routinely.

Prostate tissue (Zaichick S and Zaichick V, INAA application in the agedynamics assessment of Br, Ca, Cl, K, Mg, Mn, and Na content in thenormal human prostate. J Radioanal Nucl Chem 2011; 288:197-202),expressed prostatic secretions (EPS) (Costello L and Franklin R.Prostatic fluid electrolyte composition for the screening of prostatecancer: a potential solution to a major problem. Prostate CancerProstatic Dis 2008; 12(1): 17-24) and seminal fluid (Owen D. and Katz D.A Review of the Physical and Chemical Properties of Human Semen and theFormulation of a Semen Simulant. J Androl 2005; 26: 459-469) containunusually high amounts of electrolytes such as K, Na, Zn, Ca, Mg, Cl, Brand others. The reason for the unusually high metal ion content innormal prostate gland and its excretions is not completely understood,but it was shown that decrease in zinc levels in prostate tissue(Zaichick V, et al. SU997281), prostatic fluid (Zaichick V, et al. Zincconcentration in human prostatic fluid: normal, chronic prostatitis,adenoma and cancer. Int Urol Nephrol 1996; 28(5): 687-694) and seminalfluid (Frederickson C. US 2004/229300 A1 and US 2010/0099195 Al; andLeslie C. Costello and Renty B. Franklin, US 2011/0046204 A1) can beused to indicate the risk of prostate cancer. Until now this method hasnot found practical application.

Thus, so far no reliable method has been developed for prostate cancerdetection. Therefore there is a need for a rapid and non-invasiveroutine prostate cancer test, which can detect PCa in asymptomatic menor discriminate between benign and malignant prostatic conditions inpatients with prostatic symptoms.

SUMMARY OF THE INVENTION

The invention provides a method of diagnosing a prostate condition, asdefined in the independent claims, to which reference should now bemade. Advantageous or preferred features are set forth in dependentclaims.

According to an aspect of the invention, there may be provided a methodof diagnosing a prostate condition in a subject, comprising:

-   -   determining, in a sample obtained from a subject, a level of at        least one constituent selected from the group consisting of Ag,        Al, Au, B, Ba, Bi, Br, Ca, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe,        Gd, Hg, Ho, K, La, Li, Mg, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb,        Sc, Se, Si, Sm, Sr, Tb, Th, TI, U, Y, Zn and Zr; and    -   comparing the level of the at least one constituent in the        sample with a control level of the same at least one        constituent,        -   in which a difference between the level of the at least one            constituent in the sample and the control level of the same            at least one constituent, is indicative of the prostate            condition.

In another aspect of the invention, there may be provided a method ofdiagnosing a prostate condition in a subject, comprising:

-   -   determining, in a sample obtained from a subject, a level of at        least one constituent (or first constituent) selected from the        group consisting of Ag, Al, Au, B, Ba, Bi, Br, Ca, Cd, Ce, Co,        Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, K, La, Li, Mg, Mn, Na, Nd,        Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, TI, U, Y,        Zn and Zr; and either        -   (a) comparing a combination of levels of a plurality of            constituents from the at least one constituent in the sample            (i.e. a sample combination) with a combination of control            levels of the same plurality of constituents (i.e. a control            combination), or        -   (b) determining a level of at least one further constituent            (or second constituent) not selected from the group            consisting of Ag, Al, Au, B, Ba, Bi, Br, Ca, Cd, Ce, Co, Cr,            Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, K, La, Li, Mg, Mn, Na, Nd,            Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, TI, U,            Y, Zn and Zr, and        -   comparing a combination of the level of the at least one            constituent and the level of the at least one further            constituent in the sample (i.e. a sample combination) with a            combination of control levels of the same at least one            constituent and the same at least one further constituent            (i.e. a control combination),    -   in which a difference between the combinations is indicative of        the prostate condition.

In a preferred embodiment, the sample is, or comprises, a bodily fluid.The bodily fluid may be blood, blood plasma, urine, prostatic fluid,expressed prostatic secretion or seminal fluid. Preferably, the bodilyfluid comprises expressed prostatic secretion or seminal fluid.

Alternatively, the sample may be, or may comprise, a bodily tissue suchas prostate tissue. The prostate tissue may be obtained by biopsy.

The at least one constituent may be selected from the group consistingof Ca, K, Mg, Ag, Al, Au, B, Ba, Bi, Br, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er,Fe, Gd, Hg, Ho, La, Li, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se,Si, Sm, Sr, Tb, Th, TI, U, Y and Zr. This group may be particularlypreferable, should the sample be a bodily fluid.

In another preferred embodiment, the at least one constituent isselected from the group consisting of Al, Ba, Bi, Ca, Cu, Fe, K, Mg, Mn,Se, Tb, Th, U, Y and Zn, or is selected from the group consisting of Al,Ba, Bi, Ca, Cu, Fe, K, Mg, Mn, Se, Tb, Th, U and Y. Either of thesegroups may be particularly preferable, should the sample be a bodilyfluid.

In an alternative embodiment the at least one constituent is selectedfrom the group consisting of Mn, Al, Ba, Bi, Ca, Mg, K, Se and Cr. Thisgroup may be particularly preferable, should the sample be a bodilyfluid.

The at least one constituent may be selected from the group consistingof Ca, K, Mg, Al, Au, B, Ba, Bi, Br, Cd, Ce, Cs, Dy, Er, Gd, Ho, La, Li,Na, Nd, Ni, P, Pb, Pr, S, Si, Sm, Sr, Tb, Th, Tl, U, Y and Zr. Thisgroup may be particularly preferable, should the sample be a tissuesample.

In another preferred embodiment, the at least one constituent, isselected from the group consisting of Al, Ba, Bi, Ca, Cu, Fe, K, Mg, Mn,Se and Zn, or is selected from the group consisting of Al, Ba, Bi, Ca,Cu, Fe, K, Mg, Mn and Se. Either of these groups may be particularlypreferable, should the sample be a tissue sample.

In an alternative embodiment, the at least one constituent is selectedfrom the group consisting of Al, Ba, Bi, Ca, Mg and Mn. This group maybe particularly preferable, should the sample be a tissue sample.

In an alternative embodiment the at least one constituent is selectedfrom the group consisting of Al, Ba, Bi, Ca, Cd, Cu, Fe, Mg, Mn and Ni.

The combination of constituents may comprise determining one or moreratios. For example, the method may comprise determining a ratio betweena first constituent and a second constituent, or between two or moreconstituents, selected from the group consisting of Ag, Al, Au, B, Ba,Bi, Br, Ca, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, K, La, Li,Mg, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th,Tl, U, Y, Zn and Zr.

In preferred embodiments, the method may comprise determining a ratio ofa constituent in relation to Ca, or in relation to Zn. Preferred ratiosmay include Ca/Ba, Ca/Fe, Mg/Al, Ca/Cu, Mg/Cu, Zn/Cu, Zn/Mn, Ca/Mn,Ca/P, Ca/Si, Ca/Sr, or Ca/Al.

Assessing combinations of constituents may comprise comparingrelationships between ratios of constituents. For example, a firstsample ratio may be calculated between a first constituent and a secondconstituent. A second sample ratio may be calculated between a firstconstituent (which may be the same or different from the firstconstituent of the first ratio) and a second constituent (which may bethe same or different from the second constituent of the first ratio).Either the first or second constituent of the second ratio will thus bedifferent from the first ratio. For example, relationships betweenratios may include multiples of two or more ratios such as(Ca/Cu)*(Mg/Cu); (Ca/Cu)*(Zn/Cu); or (Mg/Cu)*(Zn/Cu). Such relationshipsmay then be compared with relationships between control ratios of thesame constituents.

Combinations of constituents may include ratios between multiples of twoor more constituents. As an illustration, this may include(Ca*Mg*Zn)/(Al*Bi*Cu), (Ca*Mg*Zn)/(Mn*Bi*Se) or(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba).

Combinations of constituents may comprise multiplication of levels oftwo or more constituents. As an illustration, this may include(Zn*Rb)/10.

In another preferred embodiment, comparing a combination of levels ofconstituents with a combination of control levels of the sameconstituents may involve normalized levels of constituents. For example,constituents may be normalized to control or reference levels of thesame constituents. For instance, normalization may include dividing alevel of constituent with an average (such as a median or a mean) levelof the same constituent taken from normal individuals. A normalizedamount of a constituent, such as a normalized mass fraction of anelement, may be represented by _(n).

Combinations of normalized levels may be used. As an illustration, thismay include(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Sc_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Au_(n)*B_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Dy_(n)*Er_(n)*Fe_(n)*Gd_(n)*Ho_(n)*La_(n)*Li_(n)*Mn_(n)*Nd_(n)*Ni_(n)*Pb_(n)*Pr_(n)*Sb_(n)*Si_(n)*Sm_(n)*Sr_(n)*Th_(n)*Tl_(n)*U_(n)*Y_(n)*Zr_(n))or(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Li_(n)*Mn_(n)*Ni_(n)*Pb_(n)*Sb_(n)*Si_(n)*Sr_(n)).

In yet another preferred embodiment, combinations of normalized levelsof constituents may be used. As an illustration this may include the useof multiplicative indices, such as(Ca_(n)*K_(n)*Mg_(n)*Rb_(n)*S_(n)*Zn_(n))/6 or(Ca_(n)*K_(n)*Mg_(n)*Zn_(n))/4. This combination may be particularlypreferable, should the sample be a bodily fluid.

In another aspect of the invention, combinations of normalized levels ofconstituents may represent the sum of normalized levels, such asnormalized mass fractions. As an illustration an additive index, such as(Ca_(n)+K_(n)+Mg_(n)+Zn_(n))−4, may be used. This additive combinationmay be particularly preferable, should the sample be an expressedprostatic secretion.

In an embodiment of the invention, the method may include the step ofobtaining a sample from a subject. Determination of levels ofconstituents in samples from a subject may occur ex vivo or in vitro.

The at least one further (or second) constituent which is not selectedfrom the group consisting of Ag, Al, Au, B, Ba, Bi, Br, Ca, Cd, Ce, Co,Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho, K, La, Li, Mg, Mn, Na, Nd, Ni, P,Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb, Th, Tl, U, Y, Zn and Zr, maybe any chemical element or any chemical substance such as a protein, DNAor RNA, or any other gene derived product.

The prostate condition may be benign prostatic hyperplasia. Preferably,the prostate condition is prostate cancer.

In another aspect of the invention, there may be provided a device forcarrying out a method; the method as described in any form above.

One aim of the present invention may be to provide a rapid, specific,non-invasive and sensitive diagnostic method of establishing thecondition of prostatic gland, in particular early non-invasive detectionof prostate cancer.

In a broad sense, the method is based on determination of the levels ofcertain chemical elements present in a biological sample from a subjectto establish the prostate condition. The obtained levels and/or anyratio between at least one of the obtained levels and the level of anychemical element or any chemical substance such as a protein, DNA orRNA, or any other gene derived product present in the biological samplefrom the same subject, and/or any combination of said ratios or saidlevels may be compared to control levels, ratio of the control levels ortheir combination. Differential presence of the said biomarkers ascompared to the control may be indicative of the prostate condition.

In one aspect, there may be provided a device for detection of thelevels of certain chemical elements as biomarkers in a biological sampleto establish the prostate condition. The obtained levels and/or anyratio between at least one of the obtained levels and the level of anychemical element, chemical substance or protein in the biological samplefrom the same subject, and/or any combination of said ratios or saidlevels may be compared to control levels, ratio of the control levels ortheir combination. Differential presence of the said biomarkers ascompared to the control may be indicative of the prostate condition.

In another aspect, there may be provided the use of determination of thelevels of certain chemical elements as biomarkers in a biological samplefor establishing the prostate condition. The obtained levels and/or anyratio between at least one of the obtained levels and the level of anychemical element, chemical substance or protein in the biological samplefrom the same subject, and/or any combination of said ratios or saidlevels may be compared to control levels, ratio of the control levels ortheir combination. Differential presence of the said biomarkers ascompared to the control may be indicative of the prostate condition.

Comparing a level of a constituent with a control level of theconstituent, or a combination of levels of constituents with acombination of control levels of the constituents may provide anindication of the presence or absence of a prostate condition.

The method may also relate to a device or tool to establish the prostatecondition.

Definitions

As used herein, the term, “a” or “an” may mean one or more. As usedherein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one but it is alsoconsistent with the meaning of “one or more”, “at least one”, and “oneor more than one”. Some embodiments of the invention may consist of orconsist essentially of one or more elements, method steps, and/ormethods of invention. It is contemplated that any method describedherein can be implemented with respect to any other method describedherein. As used herein “another” or “other” may mean at least a secondor more of the same or different claim element or components thereof.

“Biomarker” means a chemical element that is differentially present(i.e., increased or decreased) in a biological sample from a subject ora group of subjects having a first phenotype (e.g., having a disease) ascompared to a biological sample from a subject or group of subjectshaving a second phenotype (e.g., not having the disease). A biomarker ispreferably differentially present at a level that is statisticallysignificant (i.e., a p-value less than 0.05 and/or a q-value of lessthan 0.10 as determined using either Welch's T-test or Wilcoxon'srank-sum Test).

The “level” of one or more biomarkers, or constituents, means theabsolute, relative or normalised amount or concentration of thebiomarker or constituent in the sample. As used herein “control” or“control level” indicates the level of a biomarker or constituent thatis present in a sample without a particular condition, such as a healthynon-cancerous sample, which may be a sample without the prostatecondition, or may be a sample with a benign prostate condition, such asbenign prostatic hyperplasia. Such “levels” may be tailored to specifictechniques that are used to measure levels of biomarkers, orconstituents, in biological samples (e.g., ICP-MS, ICP-AES, EXDRF,colorimetric detection, voltammetry etc.), where the levels ofbiomarkers or constituents may differ based on the specific techniquethat is used. The method may include measuring mass fraction levels ofthe constituents.

“Prostate cancer” refers to a disease in which cancer develops in theprostate, a gland in the male reproductive system.

“Benign prostatic hyperplasia” refers to a histologic diagnosischaracterized by proliferation of the cellular elements of the prostate,a gland in the male reproductive system.

“Sample” or “biological sample” means biological material isolated froma subject. The biological sample may contain any biological materialsuitable for detecting the desired biomarkers, and may comprise cellularand/or non-cellular material from the subject. The sample can beisolated from any suitable biological tissue or bodily fluid such as,for example, prostate tissue, blood, blood plasma, urine, prostaticfluid, expressed prostatic secretion or seminal fluid.

“Subject” means any animal, but is preferably a mammal, such as, forexample, a human.

“False positive” is a test result that indicates that a subject has aspecific disease or condition when the subject actually does not havethe disease or condition.

“False negative” is a test result that indicates that a subject does nothave a specific disease or condition when the subject actually has thedisease or condition.

“True positive” is a test result that indicates that a subject has aspecific disease or condition when the subject actually has the diseaseor condition.

“True negative” is a test result that indicates that a subject does nothave a specific disease or condition when the subject actually does nothave the disease or condition.

Test sensitivity is calculated using following equation:

Sensitivity={True Positives (TP)/[TP+False Negatives (FN)]}×100%

Test specificity is calculated using following equation:

Specificity={True Negatives (TN)/[TN+False Positives (FP)]}×100%

Test accuracy is calculated using following equation:

Accuracy=[(TP+TN)/(TP+FP+TN+FN)]×100%

“Combination” of levels of constituents refers to any mathematicalrelationship or manipulation between levels of two or more constituents.As described above, this may include a ratio between levels ofconstituents (or the quotient of constituents), such as Ca/Fe. It mayalso include a multiple (or product) of ratios, such as (Ca/Cu)*(Mg/Cu),or it may include a ratio (or quotient) between multiples (or products)of the levels of two or more constituents, such as(Ca*Mg*Zn)/(Al*Bi*Cu). The combination may also include the product ofthe levels of two or more constituents.

Other and further aspects, features, benefits, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings have been included herein so that the citedfeatures, advantages, and objects of the invention will become clear andcan be understood in detail. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate preferred embodiments of the invention and should not beconsidered to limit the scope of the invention.

FIG. 1 shows individual data sets for Al mass fractions (mg/kg of drytissue) in samples of normal (1), benign hyperplastic (2) and cancerousprostate tissue (3).

FIG. 2 shows individual data sets for Ba mass fractions (mg/kg of drytissue) in samples of normal (1), benign hyperplastic (2) and cancerousprostate tissue (3).

FIG. 3 shows individual data sets for Bi mass fractions (mg/kg of drytissue) in samples of normal (1), benign hyperplastic (2) and cancerousprostate tissue (3).

FIG. 4 shows individual data sets for Ca mass fractions (mg/kg of drytissue) in samples of normal (1), benign hyperplastic (2) and cancerousprostate tissue (3).

FIG. 5 shows individual data sets for Mg mass fractions (mg/kg of drytissue) in samples of normal (1), benign hyperplastic (2) and cancerousprostate tissue (3).

FIG. 6 shows individual data sets for Mn mass fractions (mg/kg of drytissue) in samples of normal (1), benign hyperplastic (2) and cancerousprostate tissue (3).

FIG. 7 shows individual data sets for Ca/Fe mass fraction ratio insamples of normal (1), benign hyperplastic (2) and cancerous prostatetissue (3).

FIG. 8 shows individual data sets for Mg/AI mass fraction ratio insamples of normal (1), benign hyperplastic (2) and cancerous prostatetissue (3).

FIG. 9 shows individual data sets for Ca/Cu mass fraction in samples ofnormal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

FIG. 10 shows individual data sets for (Ca/Cu)*(Mg/Cu) mass fractionratios combination in samples of normal (1), benign hyperplastic (2) andcancerous prostate tissue (3).

FIG. 11 shows individual data sets for (Ca/Cu)*(Zn/Cu) mass fractionratios combination in samples of normal (1), benign hyperplastic (2) andcancerous prostate tissue (3).

FIG. 12 shows individual data sets for (Mg/Cu)*(Zn/Cu) mass fractionratios combination in samples of normal (1), benign hyperplastic (2) andcancerous prostate tissue (3).

FIG. 13 shows individual data sets for Ca/Ba mass fraction ratio insamples of normal (1), benign hyperplastic (2) and cancerous prostatetissue (3).

FIG. 14 shows individual data sets for Ca/P mass fraction ratio insamples of normal (1), benign hyperplastic (2) and cancerous prostatetissue (3).

FIG. 15 shows individual data sets for Ca/Si mass fraction ratio insamples of normal (1), benign hyperplastic (2) and cancerous prostatetissue (3).

FIG. 16 shows individual data sets for Ca/Sr mass fraction ratio insamples of normal (1), benign hyperplastic (2) and cancerous prostatetissue (3).

FIG. 17 shows individual data sets for Zn/Mn mass fraction ratio insamples of normal (1), benign hyperplastic (2) and cancerous prostatetissue (3).

FIG. 18 shows individual data sets for[(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratio in samples ofnormal (1), benign hyperplastic (2) and cancerous prostate tissue (3).

FIG. 19 shows individual data sets for[(Ca*Cd*Co*Hg*K*Mg*Na*P*Rb*S*Se*Zn)/(Ag*Al*Ba*Bi*Br*Ce*Cr*Cs*Cu*Li*Mn*Ni*Pb*Sb*Si*Sr)]*10¹⁸ in samples of normal (1), benign hyperplastic (2) andcancerous prostate tissue (3).

FIG. 20 shows individual data sets for[(Ca*Cd*Co*Hg*K*Mg*Na*P*Rb*S*Sc*Se*Zn)/(Ag*Al*Au*B*Ba*Bi*Br*Ce*Cr*Cs*Cu*Dy*Er*Fe*Gd*Ho*La*Li*Mn*Nd*Ni*Pb*Pr*Sb*Si*Sm*Sr*Th*TI*U*Y*Zr)]*10³⁴ in samplesof normal (1), benign hyperplastic (2) and cancerous prostate tissue(3).

FIG. 21 shows individual data sets for normalized mass fraction additiveindices of four selected elements in samples of normal, benignhyperplastic and cancerous EPS.

FIG. 22 shows individual data sets for normalized mass fractionmultiplicative indices of four selected elements in samples of EPS fromnormal, benign hyperplastic and cancerous patients.

FIG. 23 shows individual data sets for normalized mass fractionmultiplicative indices of six selected elements in samples of EPS fromnormal, benign hyperplastic and cancerous patients.

FIG. 24. Individual data sets for product index (Rb*Zn)/10 obtained fromthe EPS samples from healthy (1), benign hypertrophic (2) and cancerousindividuals (3).

The following examples are given for the purpose of illustrating thevarious embodiments of the present invention and are not meant to limitthe present invention in any fashion.

SPECIFIC EXAMPLES Example 1 Identification of Cancer Biomarkers inProstate Tissue Using Inductively Coupled Plasma Mass Spectrometry(ICP-MS) and Inductively Coupled Atomic Emission Spectrometry (ICP-AES)

Experimental conditions of the present study were approximated to thehospital conditions as closely as possible.

Equipment:

Autoclave (Ancon-AT2, Russia), inductively coupled plasma massspectrometry instrument Thermo-Fisher “X-7” (Thermo Electron, USA),Spectrometer ICAP-61 (Thermo Jarrell Ash, USA).

Specimen:

Benign prostate hyperplasia samples (n=43) and prostate adenocarcinomasamples (n=60) were obtained by transrectal biopsy of an indurated siteof prostate. Samples of the human normal prostate tissue (n=37) wereobtained at autopsy of male patients aged 41-87 died of an injury or ina car accident. The presence or absence of cancer in tissue samples wasconfirmed by microscopic analysis of tissue morphology.

Reagents:

HNO₃ (nitric acid 65% for analysis, max. 0.005 ppm Hg, GR, ISO, Merck),H₂O₂ (hydrogen peroxide pure for analysis, Merck), ICP-MS standardsICP-MS-68A and ICP-AM-6-A (High-Purity Standards, Charleston, S.C.29423, USA), ICP stock solutions (High-Purity Standards, Charleston,S.C. 29423, USA).

Protocol:

1.5 mL of HNO₃ and 0.3 mL of H₂O₂ were added to homogenized andfreeze-dried prostate tissue sample, placed in one-chamber autoclave,and decomposed for 3 hours at 160-200° C. The heat-treated sample wascooled down to the room temperature; the soluble fraction was dilutedwith deionized water to 20 mL and transferred to a plastic measuringbottle. Simultaneously, the same procedure was performed on a samplecontaining no prostate tissue, and the resultant solution was used as ablank sample. All samples were analysed by Inductively Coupled PlasmaMass Spectrometry and Inductively Coupled Plasma Atomic EmissionSpectrometry.

The spectrometer parameters and the main parameters of ICP-MSmeasurements: generator output power 1,250 W, spray chamber cooled at 3°C., plasma gas flow rate—12 L/min, nebuliser—Polycon, auxiliary air flowrate—0.9 L/min, nebulizer flow rate—0.9 L/min, sample update—0.8 mL/min,resolution—0.8, detector mode—double, scanning mode—survey scan (numberof runs—10, dwell time—0.6 ms, channels per mass—10, acquisitionduration—13.2 s) and peak jumping (sweeps—25, dwell time—10 ms, channelsper mass—1, acquisition duration—34 s).

The spectrometer parameters for ICP-AES measurements: generator outputpower 1,200 W, reflected power<5 W, nebuliser type—angular,plasma-forming air flow rate—18 L/min, auxiliary air flow rate—0.9L/min, air flow rate into atomiser—0.6 L/min, flow rate of the analysedsample—1.5 mL/min, zone height for plasma observation—14 mm.

Results:

The content of Ag, Al, Au, B, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Gd,Hg, Ho, La, Li, Mn, Nd, Ni, Pb, Pr, Rb, Sb, Sc, Se, Si, Sm, Th, Tl, U,Y, and Zr in prostate tissue was analysed by ICP-MS. The content of Na,Mg, P, S, K, Ca, Fe, Cu, Zn, Sr, and Ba in prostate tissue was analysedby ICP-AES.

Statistically significant differences in mass fraction levels of 45chemical elements (Table 1) were found in samples derived fromcancerous, benign hyperplastic and normal prostate tissues. Differencesin mass fraction levels of these elements can be used for diagnosis andtherapeutic purpose. The data in Table 1 allow evaluating the importanceof the individual chemical element content information for the diagnosisof PCa.

TABLE 1 Comparison of mean values (M ± SEM) of chemical element massfractions (mg · kg⁻¹, dry mass basis) in normal, benign hyperplastic(BPH) and cancerous (PCa) prostate tissue Prostate tissue Ratios, p(t-test) Normal BPH PCa BPH PCa PCa 41-87 year 38-83 year 40-79 year toto to Element n = 37 n = 43 n = 60 Normal Normal BPH Ag  0.0284 ± 0.0035 0.0407 ± 0.0088   0.255 ± 0.031 1.43 8.98^(c) 6.27^(c) Al    34.1 ± 3.5   24.4 ± 3.2     328 ± 73 0.72 9.62^(c) 13.4^(c) Au  0.0041 ± 0.0008 0.0026 ± 0.0008  0.0297 ± 0.0056 0.63 7.24^(c) 11.4^(c) B    1.04 ±0.18    1.51 ± 0.26    12.6 ± 3.7 1.45 12.1^(b) 8.34^(b) Ba    1.48 ±0.21    1.22 ± 0.20    26.7 ± 7.6 0.82 18.0^(b) 21.9^(b) Bi   0.029 ±0.011   0.140 ± 0.042    1.76 ± 0.27 4.83^(a) 60.7^(c) 16.9^(c) Br   27.9 ± 2.9    30.0 ± 2.6    99.9 ± 8.9 1.08 3.58^(c) 3.33^(c) Ca   2397 ± 235    2032 ± 165     675 ± 58 0.85 0.28^(c) 0.33^(c) Cd   1.12 ± 0.12    1.07 ± 0.43   0.425 ± 0.099 0.96 0.38^(c) 0.40 Ce 0.0309 ± 0.0050  0.0128 ± 0.0019   0.101 ± 0.013 0.41^(b) 3.27^(c)7.89^(c) Co  0.0452 ± 0.0043  0.0716 ± 0.0097  0.0326 ± 0.0037 1.58^(a)0.72^(a) 0.46^(b) Cr    0.53 ± 0.08    1.07 ± 0.12    2.35 ± 0.372.02^(c) 4.43^(c) 2.20^(b) Cs  0.0339 ± 0.0033  0.0235 ± 0.0025  0.0389± 0.0039 0.69^(a) 1.14 1.66^(b) Cu    9.85 ± 0.97    9.86 ± 1.25    17.1± 2.0 1.00 1.74^(b) 1.74^(b) Dy  0.0029 ± 0.0005  0.0016 ± 0.0002 0.0072 ± 0.0011 0.53^(a) 2.48^(c) 4.50^(c) Er 0.00148 ± 0.00023 0.00072± 0.00013 0.00297 ± 0.00038 0.49^(b) 2.01^(b) 4.13^(c) Fe     111 ± 9    139 ± 10     165 ± 15 1.25 1.49^(a) 1.19 Gd  0.0029 ± 0004  0.0015 ±0.0003  0.0094 ± 0.0017 0.52^(b) 3.24^(c) 6.27^(c) Hg   0.052 ± 0.009  0.275 ± 0.036   0.130 ± 0.021 5.29^(c) 2.50^(c) 0.47^(b) Ho 0.00057 ±0.00008 0.00032 ± 0.00005 0.00178 ± 0.00022 0.56^(a) 3.12^(c) 5.56^(c) K  12030 ± 475   14472 ± 740    8542 ± 504 1.20^(b) 0.71^(c) 0.59^(c) La  0.080 ± 0.019   0.019 ± 0.003   0.970 ± 0.540 0.24^(b) 12.1 51.1 Li 0.0419 ± 0.0055  0.0385 ± 0.0073   0.251 ± 0.054 0.92 5.99^(b) 6.52^(a)Mg    1071 ± 76    1201 ± 83     346 ± 61 1.12 0.32^(c) 0.29^(c) Mn   1.32 ± 0.08    1.19 ± 0.09    6.99 ± 1.35 0.90 5.30^(c) 5.87^(c) Na  10987 ± 394   11612 ± 869    7511 ± 643 1.06 0.68^(c) 0.65^(c) Nd 0.0137 ± 0.0021  0.0062 ± 0.0009  0.0413 ± 0.0065 0.45^(b) 3.01^(c)6.66^(c) Ni    3.10 ± 0.51    3.22 ± 1.06    6.96 ± 1.04 1.04 2.25^(c)2.16^(b) P    7617 ± 368    7907 ± 418    6675 ± 465 1.04 0.88 0.84 Pb   2.39 ± 0.56    0.69 ± 0.16    1.81 ± 0.35 0.29^(a) 0.76 2.62^(b) Pr 0.0035 ± 0.0005  0.0015 ± 0.0003  0.0097 ± 0.0017 0.43^(b) 2.77^(b)6.47^(c) Rb    14.8 ± 0.9    14.4 ± 0.7     8.8 ± 0.7 0.97 0.59^(c)0.61^(c) S    8557 ± 254    8787 ± 487    5343 ± 389 1.03 0.62^(c)0.61^(c) Sb   0.037 ± 0.005   0.142 ± 0.036   0.501 ± 0.062 3.84^(b)13.5^(c) 3.53^(c) Sc  0.0294 ± 0.0053  0.0257 ± 0.0040  0.0116 ± 0.00150.87 0.39^(b) 0.45^(b) Se   0.696 ± 0.044   1.243 ± 0.079   0.576 ±0.078 1.79^(c) 0.83 0.46^(c) Si     102 ± 11     141 ± 24     284 ± 391.38 2.78^(c) 2.02^(b) Sm  0.0027 ± 0.0004  0.0014 ± 0.0004  0.0095 ±0.0029 0.52^(a) 3.52^(a) 6.71^(b) Sr    2.34 ± 0.38    3.69 ± 0.45   5.75 ± 0.60 1.58^(a) 2.46^(c) 1.56^(a) Th  0.0034 ± 0.0007  0.0018 ±0.0003  0.0490 ± 0.0120 0.52^(a) 14.4^(c) 27.2^(c) Tl  0.0014 ± 0.0002 0.0020 ± 0.0006  0.0219 ± 0.0056 1.43 15.6^(c) 11.0^(b) U  0.0070 ±0.0021  0.0021 ± 0.0009  0.0068 ± 0.0013 0.30^(a) 0.97 3.24^(b) Y 0.0186 ± 0.0042  0.0071 ± 0.0012  0.0340 ± 0.0038 0.38^(a) 1.83^(b)4.79^(c) Zn    1061 ± 153    1136 ± 96     136 ± 9.9 1.07 0.13^(c)0.12^(c) Zr   0.036 ± 0.006   0.091 ± 0.036    2.13 ± 0.89 2.53 59.2^(a)23.4^(a) M-arithmetic mean, SEM-standard error of mean, ^(a)p ± 0.05,^(b)p ± 0.01, ^(c)p ± 0.001.

Example 2 Establishing the Prostate Condition using Al Mass Fraction inProstate Tissue Sample

The tissue content of Al was found to be significantly different in mostcancerous prostate tissues as compared to normal and benign hyperplastictissues (Example 1, Table 1). Mass fraction of Al in tissue of normalprostate was found to be 34.1±3.5 (SEM) mg/kg, in BPH 24.4±3.2 (SEM)mg/kg, and in PCa 328±73 (SEM) mg/kg on dry mass basis (Table 1). Theupper limit for Al mass fraction in dry normal and BPH prostate tissuewas determined to be M+2SD or 70 mg/kg on dry mass basis (FIG. 1).

If PCa needs to be discriminated from normal and BPH tissue and if Alcontent in a prostate biopsy sample prepared and analyzed as describedin the Example 1 exceeds 70 mg/kg dry tissue, prostate carcinoma with anaccuracy of 82±12% can be diagnosed. The sensitivity and specificity ofthe Al based test is 97±3% and 94±4%, respectively.

Example 3 Establishing the Prostate Condition Using Ba Mass Fraction inProstate Tissue Sample

The tissue content of Ba was found to be significantly different in mostcancerous prostate tissues as compared to normal and benign hyperplastictissues (Example 1, Table 1). Mass fraction of Ba in tissue of normalprostate was found to be 1.48±0.21 (SEM) mg/kg, in BPH 1.22±0.20 (SEM)mg/kg, and in PCa 26.7±7.6 (SEM) mg/kg on dry mass basis (Table 1). Theupper limit for Ba mass fraction in dry normal and BPH prostate tissuewas determined to be M+2SD or 3.5 mg/kg on dry mass basis (FIG. 2).

If PCa needs to be discriminated from normal and BPH tissue and if Bacontent in a prostate biopsy sample prepared and analyzed as describedin the Example 1 exceeds 3.5 mg/kg dry tissue, prostate carcinoma withan accuracy of 82±12% can be diagnosed. The sensitivity and specificityof the Ba based test is 97±3% and 94±4%, respectively.

Example 4 Establishing the Prostate Condition Using Bi Mass Fraction inProstate Tissue Sample

The tissue content of Bi was found to be significantly different in mostcancerous prostate tissues as compared to normal and benign hyperplastictissues (Example 1, Table 1). Mass fraction of Bi in tissue of normalprostate was found to be 0.029±0.011 (SEM) mg/kg, in BPH 0.140±0.042(SEM) mg/kg, and in PCa 1.76±0.27 (SEM) mg/kg on dry mass basis (Table1). The upper limit for Bi mass fraction in dry normal and BPH prostatetissue was determined to be M+2SD or 0.5 mg/kg on dry mass basis (FIG.3).

If PCa needs to be discriminated from normal and BPH tissue and if Bicontent in a prostate biopsy sample prepared and analyzed as describedin the Example 1 exceeds 0.5 mg/kg dry tissue, prostate carcinoma withan accuracy of 82±12% can be diagnosed. The sensitivity and specificityof the Bi based test is 97±3% and 93±4%, respectively.

Example 5 Establishing the Prostate Condition Using Ca Mass Fraction inProstate Tissue Sample

The tissue content of Ca was found to be significantly different in mostcancerous prostate tissues as compared to normal and benign hyperplastictissues (Example 1, Table 1). Mass fraction of Ca in tissue of normalprostate was found to be 2397±235 (SEM) mg/kg, in BPH 2032±165 (SEM)mg/kg, and in PCa 675±58 (SEM) mg/kg on dry mass basis (Table 1). Theupper limit for Ca mass fraction in dry cancerous prostate tissue wasdetermined to be M+2SD or 1080 mg/kg on dry mass basis (FIG. 4).

If PCa needs to be discriminated from normal and BPH tissue and if Cacontent in a prostate biopsy sample prepared and analysed as describedin the Example 1 does not exceed 1080 mg/kg dry tissue, prostatecarcinoma with an accuracy of 98% can be diagnosed. The sensitivity andspecificity of the Ca based test is 98% and 97%, respectively.

Example 6 Establishing the Prostate Condition Using Mg Mass Fraction inProstate Tissue Sample

The tissue content of Mg was found to be significantly different in mostcancerous prostate tissues as compared to normal and benign hyperplastictissues (Example 1, Table 1). Mass fraction of Mg in tissue of normalprostate was found to be 1071±7 (SEM) mg/kg, in BPH 1201±83 (SEM) mg/kg,and in PCa 346±61 (SEM) mg/kg on dry mass basis (Table 1). The upperlimit for Mg mass fraction in dry cancerous prostate tissue wasdetermined to be M+2SD or 700 mg/kg on dry mass basis (FIG. 5).

If PCa needs to be discriminated from normal and BPH tissue and if Mgcontent in a prostate biopsy sample prepared and analysed as describedin the Example 1 does not exceed 700 mg/kg dry tissue, prostatecarcinoma with an accuracy of 90±4% can be diagnosed. The sensitivityand specificity of the Mg based test is 100-10% and 84±6%, respectively.

Example 7 Establishing the Prostate Condition Using Mn Mass Fraction inProstate Tissue Sample

The tissue content of Mn was found to be significantly different in mostcancerous prostate tissues as compared to normal and benign hyperplastictissues (Example 1, Table 1). Mass fraction of Mn in tissue of normalprostate was found to be 1.32±0.08 (SEM) mg/kg, in BPH 1.19±0.09 (SEM)mg/kg, and in PCa 6.99±1.35 (SEM) mg/kg on dry mass basis (Table 1). Theupper limit for Mn mass fraction in dry normal or BPH prostate tissuewas determined to be M+2SD or 2 mg/kg on dry mass basis (FIG. 6).

If PCa needs to be discriminated from normal and BPH tissue and if Mncontent in a prostate biopsy sample prepared and analysed as describedin the Example 1 exceeds 2 mg/kg dry tissue, prostate carcinoma with anaccuracy of 96±3% can be diagnosed. The sensitivity and specificity ofthe Mn based test is 91±9% and 97±3%, respectively.

Example 8 Determination of Mass Fraction Levels of 44 Elements Relativeto the Mass Fraction of Calcium in Normal, Cancerous and BPH ProstateTissue

Mass fraction ratios of the elements mentioned in the Example 1 aredifferent in non-cancerous and cancerous tissue and therefore these canbe used as prostate tumor biomarkers. In the Table 2 mass fractionratios of 44 elements relative to mass fraction of calcium arepresented. Further, ratios of the mass fraction ratios for normalprostate tissue, BPH and cancerous tissue are given. The mass fractionratios presented in the Table 2 is a mean of ratios calculated for everysingle prostate sample. The data in the Table 2 allow evaluating theimportance of individual mass fraction ratios of 44 elements relative tothe mass fraction of calcium for the diagnosis of PCa.

TABLE 2 Means of ratios (M ± SEM), their ratios and the reliability ofdifference between mean values of mass fraction ratios of Ca to massfractions of other chemical element in normal, benign hyperplastic (BPH)and cancerous (PCa) prostate tissue. Prostate tissue Ratios of means, p(-test) Mass Normal BPH PCa BPH PCa PCa fraction 41-79 year 38-83 year40-79 year to to to ratio n = 37 n = 43 n = 60 Normal Normal BPH Ca/Ag  107037 ± 15763   117550 ± 34515     4164 ± 1611 1.10 0.039^(c)0.035^(b) Ca/Al      103 ± 21      101 ± 18     5.24 ± 1.9 0.980.051^(c) 0.052^(c) Ca/B     4320 ± 805     1550 ± 191      119 ± 580.36^(b) 0.028^(c) 0.077^(c) Ca/Ba     2957 ± 577     2034 ± 251     102 ± 47 0.69 0.035^(c) 0.050^(c) Ca/Bi   532698 ± 114578    93934± 41193     7501 ± 6139 0.18^(c) 0.014^(c) 0.080^(a) Ca/Br     91.3 ±12.7     78.9 ± 14.6     11.1 ± 4.8 0.86 0.12^(c) 0.14^(c) Ca/Cd    3085 ± 455     3753 ± 732     2002 ± 212 1.22 0.65^(a) 0.53^(a)Ca/Ce   144087 ± 28909   191735 ± 31186     8862 ± 2222 1.33 0.062^(c)0.046^(c) Ca/Co    69329 ± 11034    42314 ± 4982    13669 ± 10720.61^(a) 0.20^(c) 0.32^(c) Ca/Cr    14516 ± 6572     2169 ± 218      191± 41 0.15 0.013^(a) 0.088^(c) Ca/Cs    94882 ± 17335    92843 ± 9485   22723 ± 6474 0.98 0.24^(c) 0.24^(c) Ca/Cu      315 ± 47      223 ± 23      56 ± 14 0.71 0.18^(c) 0.25^(c) Ca/Dy  1733952 ± 444593  1685620 ±327920   161389 ± 47689 0.97 0.093^(c) 0.096^(c) Ca/Er  2782727 ± 557202 3989832 ± 845199   296667 ± 64924 1.43 0.11^(c) 0.074^(c) Ca/Fe    20.8 ± 2.4     17.8 ± 1.9     4.81 ± 0.53 0.86 0.23^(c) 0.27^(c)Ca/Gd  1489869 ± 334486  1726552 ± 327682   118454 ± 35677 1.160.080^(c) 0.069^(c) Ca/Hg    78186 ± 11882     9944 ± 1259     4445 ±2330 0.13^(c) 0.057^(c) 0.45^(a) Ca/Ho  7482530 ± 1547065  8358623 ±1644445   453653 ± 88284 1.12 0.061^(c) 0.054^(c) Ca/K    0.227 ± 0.037   0.144 ± 0.013    0.081 ± 0.008 0.63^(a) 0.36^(c) 0.56^(c) Ca/La  105705 ± 19452   128328 ± 16885     7340 ± 3488 1.21 0.069^(c)0.057^(c) Ca/Li    79700 ± 10834    71782 ± 11904     5847 ± 2025 0.900.073^(c) 0.082^(c) Ca/Mg     2.83 ± 0.51     1.72 ± 0.12     2.58 ±0.47 0.61 0.91 1.50 Ca/Mn     2061 ± 325     1789 ± 186      181 ± 660.87 0.088^(c) 0.10^(c) Ca/Na    0.236 ± 0.032    0.189 ± 0.025    0.097± 0.014 0.80 0.41^(c) 0.51^(b) Ca/Ni     1916 ± 626     1028 ± 179     123 ± 28 0.54 0.064^(b) 0.12^(c) Ca/P    0.348 ± 0.040    0.264 ±0.025    0.112 ± 0.020 0.76 0.32^(c) 0.42^(c) Ca/Pb     3774 ± 724    4461 ± 756      556 ± 149 1.18 0.15^(c) 0.12^(c) Ca/Pr  1263853 ±269288  2231480 ± 735010   112525 ± 35218 1.77 0.089^(c) 0.050^(b) Ca/Rb     180 ± 23      139 ± 12     81.8 ± 7.8 0.77 0.45^(c) 0.59^(c) Ca/S   0.308 ± 0.045    0.238 ± 0.022    0.133 ± 0.016 0.77 0.43^(c)0.56^(c) Ca/Sb   121963 ± 24741    49028 ± 20319     2784 ± 556 0.40^(a)0.023^(c) 0.057^(a) Ca/Sc   174958 ± 51707    76930 ± 10995    49945 ±6858 0.44 0.29^(a) 0.65^(a) Ca/Se     3604 ± 500     2362 ± 296      895± 168 0.66^(a) 0.25^(c) 0.38^(c) Ca/Si     35.7 ± 6.9     17.4 ± 2.3    3.21 ± 0.88 0.49^(a) 0.090^(c) 0.18^(c) Ca/Sm  1695658 ± 438262 2939100 ± 810027   142073 ± 37596 1.73 0.084^(b) 0.048^(c) Ca/Sr    1334 ± 142      743 ± 76      137 ± 34 0.56^(b) 0.10^(c) 0.18^(c)Ca/Th  1703628 ± 375869  1499284 ± 275745    43707 ± 21300 0.880.026^(c) 0.029^(c) Ca/Tl  2870569 ± 627543  1464103 ± 252751   124174 ±74903 0.51^(a) 0.043^(c) 0.085^(c) Ca/U  1122953 ± 182815  2061625 ±434930   130793 ± 22073 1.84 0.12^(c) 0.063^(c) Ca/Y   414438 ± 116105  385834 ± 74931    23034 ± 3863 0.93 0.056^(b) 0.060^(c) Ca/Zn     3.89± 0.91     1.72 ± 0.21     5.02 ± 0.41 0.44^(a) 1.29 2.92^(c) Ca/Zr  135701 ± 31300    61766 ± 18949      853 ± 238 0.46^(a) 0.0063^(c)0.014^(c) M-arithmetic mean, SEM-standard error of mean, ^(a)p ≤ 0.05,^(b)p ≤ 0.01, ^(c)p ≤ 0.001.

Example 9 Determination of Mass Fraction Levels of 44 Elements Relativeto Mass Fraction of Zinc in Normal, Cancerous and BPH Prostate Tissue

Mass fraction ratios of the elements mentioned in the Example 1 aredifferent in non-cancerous and cancerous tissue and therefore these canbe used as prostate tumor biomarkers. In the Table 3 mass fractionratios of 44 elements relative to mass fraction of zinc are presented.Further, ratios of the mass fraction ratios for normal prostate tissue,BPH and cancerous tissue are given. The mass fraction ratios presentedin the Table 3 is a mean of ratios calculated for every single prostatesample. The data in the Table 3 allow evaluating the importance ofindividual mass fraction ratios of 44 elements relative to the massfraction of Zn for the diagnosis of PCa.

TABLE 3 Means of ratios (M ± SEM), their ratios and the reliability ofdifference between mean values of mass fraction ratios of Zn to massfractions of other chemical element in normal, benign hyperplastic (BPH)and cancerous (PCa) prostate tissue Prostate tissue Ratios, p (t-test)Mass Normal BPH PCa BPH PCa PCa fraction 41-79 year 38-83 year 40-79year to to to ratio n = 37 n = 43 n = 60 Normal Normal BPH Zn/Ag   32271 ± 5360    39748 ± 4328      723 ± 133 1.23 0.022^(c) 0.018^(c)Zn/Al     41.3 ± 9.7     59.0 ± 9.8     1.16 ± 0.52 1.43 0.028^(c)0.020^(c) Zn/Au   645790 ± 240530   816590 ± 173610    19120 ± 122101.27 0.029^(b) 0.023^(c) Zn/B     1974 ± 559     1360 ± 417     28.8 ±21.0 0.69 0.015^(b) 0.021^(b) Zn/Ba     1003 ± 195     1373 ± 203      30 ± 17 1.37 0.030^(c) 0.022^(c) Zn/Bi   236160 ± 62300    79960 ±37890     2290 ± 2110 0.34^(a) 0.0097^(c) 0.029^(a) Zn/Br     39.1 ± 6.2    68.8 ± 11.5     1.30 ± 0.14 1.76^(a) 0.033^(c) 0.019^(c) Zn/Ca   0.449 ± 0.059    0.758 ± 0.171    0.169 ± 0.027 1.69 0.38^(c)0.22^(b) Zn/Cd    39170 ± 11900    39100 ± 6460      319 ± 59 1.000.0082^(c) 0.0082^(c) Zn/Ce    60330 ± 14060   131210 ± 22300     2055 ±899 2.17^(b) 0.035^(c) 0.016^(c) Zn/Co    27011 ± 3716    20798 ± 3359    4293 ± 554 0.77 0.16^(c) 0.21^(c) Zn/Cr     2654 ± 356     1161 ±156     78.1 ± 13.4 0.44^(c) 0.029^(c) 0.067^(c) Zn/Cs    37990 ± 8990   69050 ± 15160     3899 ± 1158 1.82 0.103^(c) 0.057^(c) Zn/Cu      114± 19      133 ± 19      9.0 ± 2.3 1.17 0.079^(c) 0.068^(c) Zn/Dy  657590 ± 148330  1194200 ± 255540    30310 ± 10770 1.81 0.046^(c)0.025^(c) Zn/Er  1190700 ± 293660  2796660 ± 590750    54040 ± 157202.35^(a) 0.045^(c) 0.019^(c) Zn/Fe      8.8 ± 1.4     11.8 ± 1.5    0.97 ± 0.11 1.34 0.11^(c) 0.082^(c) Zn/Gd   624740 ± 158040  1190620± 240260    23210 ± 8250 1.91 0.037^(c) 0.019^(c) Zn/Hg    27011 ± 3717    6490 ± 688     1216 ± 115 0.24^(c) 0.045^(c) 0.19^(c) Zn/Ho  3128380± 766220  5591120 ± 958640    97340 ± 37950 1.79 0.031^(c) 0.017^(c)Zn/K    0.086 ± 0.016    0.109 ± 0.024   0.0135 ± 0.0026 1.27 0.16^(c)0.12^(c) Zn/La    61550 ± 25120    96666 ± 23120     2156 ± 1250 1.570.035^(a) 0.022^(c) Zn/Li    35526 ± 9597    51562 ± 11566     1248 ±528 1.45 0.035^(b) 0.024^(c) Zn/Mg     1.01 ± 0.14     1.33 ± 0.35    0.38 ± 0.50 1.32 0.38 0.29 Zn/Mn      847 ± 210     1261 ± 185      43 ± 23 1.49 0.051^(c) 0.034^(c) Zn/Na    0.099 ± 0.019    0.144 ±0.035   0.0153 ± 0.0025 1.45 0.15^(c) 0.11^(b) Zn/Nd   133860 ± 32890  275460 ± 44660     4690 ± 1810 2.05^(a) 0.035^(c) 0.017^(c) Zn/Ni     712 ± 185      820 ± 220       26 ± 11 1.15 0.037^(c) 0.032^(c)Zn/P    0.128 ± 0.016    0.198 ± 0.041   0.0187 ± 0.0042 1.55 0.15^(c)0.094^(c) Zn/Pb     1523 ± 348     2910 ± 470      128 ± 58 1.91^(a)0.084^(c) 0.044^(c) Zn/Pr   529125 ± 130900  1429530 ± 348740    21630 ±7700 2.70^(a) 0.041^(c) 0.015^(c) Zn/Rb     71.7 ± 9.0     87.4 ± 9.3    17.9 ± 2.0 1.22 0.26^(c) 0.22^(c) Zn/S    0.123 ± 0.025    0.182 ±0.041   0.0213 ± 0.0035 1.48 0.17^(c) 0.12^(c) Zn/Sb    34333 ± 6156   10115 ± 2344      334 ± 44 0.29^(c) 0.0097^(c) 0.033^(c) Zn/Sc   46794 ± 7866    39678 ± 3372    13157 ± 1624 0.85 0.28^(c) 0.33^(c)Zn/Se     1548 ± 166      886 ± 90      270 ± 28 0.57^(b) 0.18^(c)0.30^(c) Zn/Si     15.8 ± 4.9     14.3 ± 4.6     0.69 ± 0.33 0.910.044^(c) 0.048^(b) Zn/Sm   642630 ± 151110  1796120 ± 413200    27835 ±9730 2.79^(a) 0.043^(c) 0.015^(c) Zn/Sr      561 ± 83      641 ± 223    22.2 ± 4.7 1.14 0.040^(c) 0.035^(b) Zn/Th   721180 ± 196050  1041050± 219340    12010 ± 7530 1.44 0.017^(c) 0.012^(c) Zn/Tl   855140 ±116010   877340 ± 132760    35010 ± 26220 1.03 0.041^(c) 0.040^(c) Zn/U  461270 ± 98980  1514290 ± 378280    23375 ± 6170 3.28^(b) 0.050^(c)0.015^(c) Zn/Y   171540 ± 52620   271240 ± 54800     4540 ± 1495 1.580.026^(b) 0.017^(c) Zn/Zr    49100 ± 9570    41930 ± 11960      151 ± 580.85 0.0031^(c) 0.0036^(c) M-arithmetic mean, SEM-standard error ofmean, ^(a)p ≤ 0.05, ^(b)p ≤ 0.01, ^(c)p ≤ 0.001.

Example 10 Using the Ca/Fe Mass Fraction Ratio to Establish ProstateCondition

The Ca/Fe mass fraction ratio was found to be significantly different inmost cancerous prostate tissues as compared to normal and benignhyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Femass fraction ratio on dry mass basis in cancerous prostate tissue wasdetermined to be M+3SD (M—arithmetic mean, SD—standard deviation) or 10(FIG. 7).

If PCa needs to be discriminated from normal and BPH tissue and if theCa/Fe ratio in a prostate biopsy sample prepared and analysed asdescribed in Example 1 does not exceed 10, prostate carcinoma with anaccuracy of 96±3% can be diagnosed. The sensitivity and specificity ofthe Ca/Fe ratio based test is 100-9% and 95±4%, respectively.

Example 11 Using the Mg/Al Mass Fraction Ratio to Establish ProstateCondition

The Mg/Al mass fraction ratio was found to be significantly different inmost cancerous prostate tissues as compared to normal and benignhyperplastic tissues. The upper limit for Mg/Al mass fraction ratio ondry mass basis in cancerous prostate tissue was determined to be M+2SD(M—arithmetic mean, SD—standard deviation) or 9 (FIG. 8).

If PCa needs to be discriminated from normal and BPH tissue and if theMg/Al ratio in a prostate biopsy sample prepared and analysed asdescribed in Example 1 does not exceed 9, prostate carcinoma with anaccuracy of 99% can be diagnosed. The sensitivity and specificity of theMg/Al ratio based test is 98% and 99%, respectively.

Example 12 Using the Ca/Cu Mass Fraction Ratio to Establish ProstateCondition

The Ca/Cu mass fraction ratio was found to be significantly different inmost cancerous prostate tissues as compared to normal and benignhyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Cumass fraction ratio on dry mass basis in cancerous prostate tissue wasdetermined to be M+3SD (M—arithmetic mean, SD—standard deviation) or 100(FIG. 9).

If PCa needs to be discriminated from normal and BPH tissue and if theCa/Cu ratio in a prostate biopsy sample prepared and analysed asdescribed in Example 1 does not exceed 100, prostate carcinoma with anaccuracy of 97±3% can be diagnosed. The sensitivity and specificity ofthe Ca/Cu ratio based test is 91±9% and 100-3%, respectively.

Example 13 Using the (Ca/Cu)*(Mg/Cu) Mass Fraction Ratio Combination toEstablish Prostate Condition

The (Ca/Cu)*(Mg/Cu) mass fraction ratio combination was found to besignificantly different in most cancerous prostate tissues as comparedto normal and benign hyperplastic tissues. The upper limit for(Ca/Cu)*(Mg/Cu) mass fraction ratio combination on dry mass basis incancerous prostate tissue was determined to be M+3SD (M—arithmetic mean,SD—standard deviation) or 4000 (FIG. 10).

If PCa needs to be discriminated from normal and BPH tissue and if the(Ca/Cu)*(Mg/Cu) ratio in a prostate biopsy sample prepared and analysedas described in Example 1 does not exceed 4000, prostate carcinoma withan accuracy of 100-2% can be diagnosed. The sensitivity and specificityof the (Ca/Cu)*(Mg/Cu) ratio based test is 100-11% and 100-3%,respectively.

Example 14 Using the (Ca/Cu)*(Zn/Cu) Mass Fraction Ratio Combination toEstablish Prostate Condition

The (Ca/Cu)*(Zn/Cu) mass fraction ratio combination was found to besignificantly different in most cancerous prostate tissues as comparedto normal and benign hyperplastic tissues. The upper limit for(Ca/Cu)*(Zn/Cu) mass fraction ratio on dry mass basis in cancerousprostate tissue was determined to be M+3SD (M—arithmetic mean,SD—standard deviation) or 1700 (FIG. 11).

If PCa needs to be discriminated from normal and BPH tissue and if the(Ca/Cu)*(Zn/Cu) ratio in a prostate biopsy sample prepared and analysedas described in Example 1 does not exceed 1700, prostate carcinoma withan accuracy of 100-2% can be diagnosed. The sensitivity and specificityof the (Ca/Cu)*(Zn/Cu) ratio based test is 100-10% and 100-3%,respectively.

Example 15 Using the (Mg/Cu)*(Zn/Cu) Mass Fraction Ratio Combination toEstablish Prostate Condition

The (Mg/Cu)*(Zn/Cu) mass fraction ratio was found to be significantlydifferent in most cancerous prostate tissues as compared to normal andbenign hyperplastic tissues. The upper limit for (Mg/Cu)*(Zn/Cu) massfraction ratio on dry mass basis in cancerous prostate tissue wasdetermined to be M+3SD (M—arithmetic mean, SD—standard deviation) or 975(FIG. 12).

If PCa needs to be discriminated from normal and BPH tissue and if the(Mg/Cu)*(Zn/Cu) ratio in a prostate biopsy sample prepared and analysedas described in Example 1 does not exceed 975, prostate carcinoma withan accuracy of 100-2% can be diagnosed. The sensitivity and specificityof the (Mg/Cu)*(Zn/Cu) ratio based test is 100-11% and 100-3%,respectively.

Example 16 Using Bodily Fluids and Tissues to Establish ProstateCondition

Using the method of analysis described in the Example 1 the massfraction ratios of Ca and Mg were determined in main histologicalcompartments of the prostate tissue: glandular epithelium, stroma andlumen. The correlation coefficients (r-value) between the mass fractionof the element in a prostate tissue compartment and the relative volumeof the main histological compartments of prostate tissue are given inthe Table 4. For Ca and Mg a strong correlation with lumen was foundindicating that the content of given markers is reflected in theprostatic fluid, which is the main part of the content of the prostatetissue lumen. Prostatic fluid is the part of the ejaculate and ispresent in urine too; therefore the concentration of the specificbiomarkers will also be reflected in ejaculate and urine. As a result,prostate condition can be established using the biomarkers given in theTable 1 using prostatic fluid, seminal fluid and urine samples.

TABLE 4 Correlation coefficient (r-value) between the mass fraction ofCa and Mg in prostate tissue and the relative volume of the mainhistological compartments of prostate tissue. Compartment/Element Ca MgGlandular epithelium 0.194 0.385 Stroma -0.421 -0.482 Lumen 0.582 0.437Statistically significant r-values are given in bold

Example 17 Identification of Cancer Biomarkes in Expressed ProstaticSecretion Using Inductively Coupled Plasma Mass Spectrometry (ICP-MS)and Inductively Coupled Atomic Emission Spectrometry (ICP-AES)

Experimental conditions of the present study were approximated to thehospital conditions as closely as possible.

Equipment:

Inductively coupled plasma mass spectrometry instrument Agilent 7500c.

Specimen:

Expressed Prostatic Secretion samples (EPS) from patients with BenignProstate Hyperplasia (BPH) and prostate adenocarcinoma (PCa) and EPSsamples from healthy volunteers were obtained by transrectal prostatemassage. The presence of cancer was confirmed by Digital RectalExamination (DRE), TransRectal Ultrasound Imaging (TRUSI) andmicroscopic analysis of tissue morphology in biopsies obtained from thesame patients. The absence of cancer was confirmed by DRE and TRUSI.

Reagents:

HNO₃ (nitric acid 65% for analysis, max. 0.005 ppm Hg, GR, ISO, Merck),H₂O₂ (hydrogen peroxide pure for analysis, Merck), ICP-MS standardsNCSZC73013 (NCS Certified Reference Material), BCR063R (Community Bureauof Reference of the European Comission) and IRM-BD151 (LGC Standards,Weisel, Germany).

Protocol:

0.5 mL of HNO₃ was added to freeze-dried EPS samples and the sampleswere left over night at room temperature. After that 0.25 mL of HNO₃ and0.15 mL of H₂O₂ were added to the samples and placed in water bath at95° C. for 30 min. The heat-treated samples were cooled down to the roomtemperature; the soluble fraction was diluted with deionized water to 15mL and transferred to a plastic measuring bottle. Simultaneously, thesame procedure was performed on a sample containing no EPS fluid, andthe resultant solution was used as a blank sample. All samples wereanalysed by Inductively Coupled Plasma Mass Spectrometry and InductivelyCoupled Plasma Atomic Emission Spectrometry.

The spectrometer parameters and the main parameters of ICP-MSmeasurements: auxiliary air flow rate—0.9 L/min, nebulizer flow rate—0.9L/min, sample update—0.8 mL/min. The spectrometer parameters for ICP-AESmeasurements: generator output power 1,500 W.

Results:

The content of Ag, Al, Au, B, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Gd,Hg, Ho, La, Li, Mn, Nd, Ni, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Tb, Th,Tl, U, Y, and Zr in EPS was analysed by ICP-MS. The content of Na, Mg,P, S, K, Ca, Fe, Cu, Zn, Sr, and Ba in EPS was analysed by ICP-AES.

Statistically significant differences in mass fraction levels of 46chemical elements (Table 5) were found in samples derived fromcancerous, benign hyperplastic and normal prostate EPS. Differences inmass fraction levels of these elements can be used for diagnosis andtherapeutic purpose. The data in Table 5 allow evaluating the importanceof the individual chemical element content information for the diagnosisof prostate cancer (PCa).

TABLE 5 Comparison of mean values of chemical element mass fractions (mg· kg⁻¹, dry mass basis) in normal, benign hyperplastic (BPH) andcancerous (PCa) EPS. Ratios of means Prostatic fluid BPH to PCa to PCato Element Normal BPH PCa Normal Normal BPH Li 0.45 0.18 0.21 0.4 0.51.2 B 0.97 2.54 1.00 2.6 1.0 0.4 Na 45440 47804 167000 1.1 3.7 3.5 Mg5130 4549 1900 0.9 0.4 0.4 Al 4.63 12.50 43.85 2.7 9.5 3.5 Si 25.8044.33 110.00 1.7 4.3 2.5 P 1732 4844 1800 2.8 1.0 0.4 S 5469 5063 63000.9 1.2 1.2 K 37920 27525 19000 0.7 0.5 0.7 Ca 11040 10758 3800 1.0 0.30.4 Sc 0.06 0.05 0.01 1.0 0.2 0.2 Cr 0.61 0.90 23.74 1.5 39.1 26.4 Mn1.10 0.71 3.95 0.7 3.9 5.6 Fe 15.7 26.4 370.0 1.7 23.6 14.0 Co 0.03 0.050.08 1.3 2.5 1.9 Ni 0.32 4.58 7.03 14.2 21.9 1.5 Cu 8.46 13.65 14.45 1.61.7 1.1 Zn 8606 5099 2000 0.6 0.2 0.4 Se 1.56 1.45 0.10 0.9 0.1 0.1 Br31.8 43.5 559.6 1.4 17.6 12.9 Rb 52.8 32.3 23.9 0.6 0.5 0.7 Sr 2.71 2.573.15 0.9 1.2 1.2 Y 0.01 0.01 0.03 1.3 2.6 2.0 Zr 0.07 0.14 0.47 2.1 6.83.3 Ag 0.03 0.37 0.94 14.4 36.2 2.5 Sb 0.10 0.51 0.10 5.1 1.0 0.2 Cs0.08 0.08 0.10 1.1 1.3 1.2 Ba 0.35 0.35 3.31 1.0 9.4 9.4 La 0.06 0.020.04 0.4 0.8 2.3 Ce 0.01 0.02 0.08 1.2 6.2 5.1 Pr 0.06 0.01 0.02 0.2 0.42.0 Nd 0.01 0.03 0.04 2.5 3.7 1.5 Sm 0.01 0.01 0.02 1.1 2.4 2.3 Gd 0.010.01 0.04 1.2 3.6 3.0 Tb 0.01 0.01 0.01 1.0 1.4 1.4 Dy 0.01 0.01 0.021.0 1.9 1.9 Ho 0.01 0.01 0.01 1.0 1.2 1.2 Er 0.01 0.01 0.03 1.0 2.5 2.5Au 0.07 0.19 0.01 2.6 0.1 0.1 Cd 0.03 0.08 0.37 3.3 14.4 4.3 Hg 0.080.05 0.0001 0.7 0.001 0.001 Tl 0.01 0.01 0.15 0.9 13.8 15.3 Pb 0.08 0.321.03 3.7 12.2 3.3 Bi 0.02 0.01 0.40 0.6 24.5 39.5 Th 0.03 0.01 0.04 0.51.7 3.8 U 0.01 0.01 0.05 1.0 4.8 4.8

Example 18 Identification of Cancer Biomarkers in Seminal Fluid UsingInductively Coupled Plasma Mass Spectrometry (ICP-MS) and InductivelyCoupled Atomic Emission Spectrometry (ICP-AES)

Experimental conditions of the present study were approximated to thehospital conditions as closely as possible.

Equipment:

Inductively coupled plasma mass spectrometry instrument Agilent 7500c.

Specimen:

Ejaculate samples from patients with Benign Prostatic Hyperplasia,prostate adenocarcinoma and from healthy volunteers were obtained bymasturbation into a clean metal-free vial. The presence of cancer wasconfirmed by DRE, TRUSI and microscopic analysis of tissue morphology inbiopsies obtained from the same patients. The absence of cancer wasconfirmed by DRE and TRUSI.

Reagents:

HNO₃ (nitric acid 65% for analysis, max. 0.005 ppm Hg, GR, ISO, Merck),H₂O₂ (hydrogen peroxide pure for analysis, Merck), ICP-MS standardsNCSZC73013 (NCS Certified Reference Material), BCR063R (Community Bureauof Reference of the European Comission) and IRM-BD151 (LGC Standards,Weisel, Germany).

Protocol:

0.5 mL of HNO₃ was added to freeze-dried seminal fluid samples and thesamples were left over night at room temperature. After that 0.25 mL ofHNO₃ and 0.15 mL of H₂O₂ were added to the samples and placed in waterbath at 95° C. for 30 min. The heat-treated sample was cooled down tothe room temperature; the soluble fraction was diluted with deionizedwater to 15 mL and transferred to a plastic measuring bottle.Simultaneously, the same procedure was performed on a sample containingno seminal fluid, and the resultant solution was used as a blank sample.All samples were analysed by Inductively Coupled Plasma MassSpectrometry and Inductively Coupled Plasma Atomic EmissionSpectrometry.

The spectrometer parameters and the main parameters of ICP-MSmeasurements: auxiliary air flow rate—0.9 L/min, nebulizer flow rate—0.9L/min, sample update—0.8 mL/min. The spectrometer parameters for ICP-AESmeasurements: generator output power 1,500 W.

Results:

The content of Ag, Al, Au, B, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Gd,Hg, Ho, La, Li, Mn, Nd, Ni, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Tb, Th,Tl, U, Y, and Zr in seminal fluid was analysed by ICP-MS. The content ofNa, Mg, P, S, K, Ca, Fe, Cu, Zn, Sr, and Ba in seminal fluid wasanalysed by ICP-AES.

Statistically significant differences in mass fraction levels of 46chemical elements (Table 6) were found in seminal fluid samples derivedfrom cancerous, benign hyperplastic and normal subjects. Differences inmass fraction levels of these elements can be used for diagnosis andtherapeutic purpose. The data in Table 6 allow evaluating the importanceof the individual chemical element content information for the diagnosisof prostate cancer (PCa).

TABLE 6 Comparison of mean values of chemical element mass fractions (mg· kg⁻¹, dry mass basis) in normal, benign hyperplastic (BPH) andcancerous (PCa) seminal fluid. Ratios of means Seminal fluid BPH to PCato PCa to Element Normal BPH PCa Normal Normal BPH Li 0.04 0.17 0.01 4.60.32 0.1 B 0.80 4.19 1.00 5.2 1.25 0.2 Na 21489 42638 35000 2.0 1.63 0.8Mg 1100 3674 140 8.2 0.13 0.04 Al 3.82 6.06 4.69 1.6 1.23 0.8 Si 6.3129.35 11.92 4.6 1.89 0.4 P 10352 8453 15000 0.8 1.45 1.8 S 1966 47162700 2.4 1.37 0.6 K 5201 21428 3800 4.1 0.73 0.2 Ca 3034 8237 1300 5.00.43 0.2 Sc 0.05 0.04 0.01 0.9 0.22 0.2 Cr 0.41 0.55 1.20 1.3 2.91 2.2Mn 0.23 0.35 0.12 1.6 0.55 0.4 Fe 19.0 17.3 8.5 0.9 0.45 0.5 Co 0.010.03 0.01 2.9 1.00 0.3 Ni 0.22 3.27 0.10 14.6 0.45 0.03 Cu 2.16 12.670.99 5.9 0.46 0.1 Zn 731 4003 140 5.5 0.19 0.03 Se 0.68 1.41 0.36 2.10.54 0.3 Br 30.8 42.5 54.7 1.4 1.78 1.3 Rb 7.0 24.1 5.0 3.4 0.71 0.2 Sr0.45 2.28 0.50 5.0 1.09 0.2 Y 0.01 0.01 0.01 0.7 0.73 1.0 Zr 0.08 0.080.05 1.0 0.59 0.6 Ag 0.01 0.01 0.03 1.0 2.99 3.0 Sb 0.10 0.10 0.01 1.00.10 0.1 Cs 0.01 0.06 0.01 4.5 0.99 0.2 Ba 0.13 0.25 0.13 1.9 1.01 0.5La 0.10 0.01 0.01 0.1 0.10 1.0 Ce 0.01 0.01 0.01 1.0 0.81 0.8 Pr 0.010.01 0.01 1.0 1.00 1.0 Nd 0.01 0.01 0.01 1.0 1.00 1.0 Sm 0.01 0.01 0.001.0 0.16 0.2 Gd 0.01 0.01 0.01 1.0 1.00 1.0 Tb 0.01 0.01 0.01 1.0 1.001.0 Dy 0.01 0.01 0.01 1.0 1.00 1.0 Ho 0.01 0.01 0.01 1.0 1.00 1.0 Er0.01 0.01 0.01 1.0 1.00 1.0 Au 0.02 0.07 0.01 3.1 0.44 0.1 Cd 0.01 0.030.02 2.1 1.58 0.7 Hg 0.04 0.05 0.01 1.4 0.01 0.2 TI 0.01 0.01 0.01 1.01.00 1.0 Pb 0.10 0.14 0.08 1.4 0.85 0.6 Bi 0.01 0.01 0.02 0.8 1.27 1.7Th 0.02 0.01 0.01 0.6 0.56 1.0 U 0.01 0.01 0.01 1.0 1.00 1.0

Example 19 Establishing the Prostate Condition Using Mn Mass Fraction inan EPS Sample

The tissue content of Mn was found to be significantly different in mostcancerous EPS samples as compared to normal and benign hyperplastic EPSsamples (Example 17, Table 5). Mass fraction of Mn in EPS of normalprostate was found to be 0.51 mg/kg, in BPH 1.10 mg/kg, and in PCa 3.95mg/kg on dry mass basis (Table 5). The upper limit for Mn mass fractionin dry EPS from normal or BPH subject was determined to be M+2SD or 2.4mg/kg on dry mass basis.

If PCa EPS needs to be discriminated from normal and BPH and if Mncontent in the EPS sample prepared and analysed as described in theExample 17 exceeds 2.4 mg/kg dry EPS, prostate carcinoma can bediagnosed with an accuracy of 94±3%.

Example 20 Establishing the Prostate Condition Using Al Mass Fraction inan EPS Sample

The tissue content of Al was found to be significantly different in mostcancerous EPS samples as compared to normal and benign hyperplastic EPSsamples (Example 17, Table 5). Mass fraction of Al in EPS of normalprostate was found to be 4.63 mg/kg, in BPH 12.5 mg/kg, and in PCa 43.85mg/kg on dry mass basis (Table 5). The upper limit for Al mass fractionin dry EPS from normal or BPH subject was determined to be M+2SD or 25mg/kg on dry mass basis.

If EPS PCa needs to be discriminated from normal and BPH and if Alcontent in a EPS sample prepared and analysed as described in theExample 17 exceeds 25 mg/kg in dry EPS, carcinoma with an accuracy of96±4% can be diagnosed.

Example 21 Establishing the Prostate Condition Using Ba Mass Fraction inan EPS Sample

The tissue content of Ba was found to be significantly different in mostcancerous prostatic fluid samples as compared to normal and benignhyperplastic prostatic fluid samples (Example 17, Table 5). Massfraction of Ba in EPS of normal prostate was found to be 0.35 mg/kg, inBPH 0.35 mg/kg, and in PCa 3.57 mg/kg on dry mass basis (Table 5). Theupper limit for Ba mass fraction in dry EPS from normal or BPH subjectwas determined to be M+2SD or 1.5 mg/kg on dry mass basis.

If EPS PCa needs to be discriminated from normal and BPH and if Bacontent in the EPS sample prepared and analysed as described in theExample 17 exceeds 2.5 mg/kg dry EPS, prostate carcinoma with anaccuracy of 95±5% can be diagnosed.

Example 22 Establishing the Prostate Condition Using Bi Mass Fraction inan EPS Sample

The tissue content of Bi was found to be significantly different in mostcancerous prostatic fluid samples as compared to normal and benignhyperplastic prostatic fluid samples (Example 17, Table 5). Massfraction of Bi in EPS of normal prostate was found to be 0.02 mg/kg, inBPH 0.01 mg/kg, and in PCa 0.40 mg/kg on dry mass basis (Table 5). Theupper limit for Bi mass fraction in dry EPS from normal or BPH subjectwas determined to be 0.04 mg/kg on dry mass basis.

If EPS PCa needs to be discriminated from normal and BPH and if Bicontent in a seminal fluid sample prepared and analysed as described inthe Example 17 exceeds 0.03 mg/kg dry EPS, prostate carcinoma with anaccuracy of 96±3% can be diagnosed.

Example 23 Establishing the Prostate Condition Using Ca Mass Fraction inan EPS Sample

The tissue content of Ba was found to be significantly different in mostcancerous prostatic fluid samples as compared to normal and benignhyperplastic prostatic fluid samples (Example 17, Table 5). Massfraction of Ca in EPS of normal prostate was found to be 11040 mg/kg, inBPH 10758 mg/kg, and in PCa 3800 mg/kg on dry mass basis (Table 5). Thelower limit for Ca mass fraction in dry EPS from normal or BPH subjectwas determined to be 8000 mg/kg on dry mass basis.

If EPS PCa needs to be discriminated from normal and BPH and if Cacontent in the EPS sample prepared and analyzed as described in theExample 17 does not exceed 2000 mg/kg dry EPS, prostate carcinoma withan accuracy of 95±5% can be diagnosed.

Example 24 Establishing the Prostate Condition Using Mg Mass Fraction inan EPS Sample

The tissue content of Mg was found to be significantly different in mostcancerous prostatic fluid samples as compared to normal and benignhyperplastic prostatic fluid samples (Example 17, Table 5). Massfraction of Mg in EPS of normal prostate was found to be 5130 mg/kg, inBPH 4549 mg/kg, and in PCa 1900 mg/kg on dry mass basis (Table 5). Thelower limit for Mg mass fraction in dry EPS from normal or BPH subjectwas determined to be 3500 mg/kg on dry mass basis.

If EPS PCa needs to be discriminated from normal and BPH and if mgcontent in the EPS sample prepared and analysed as described in theExample 17 does not exceed 3500 mg/kg dry EPS, prostate carcinoma withan accuracy of 95±5% can be diagnosed.

Example 25 Establishing the Prostate Condition Using Cr Mass Fraction ina Seminal Fluid Sample

The tissue content of Cr was found to be significantly different in mostcancerous seminal fluid samples as compared to normal and benignhyperplastic seminal fluid samples (Example 18, Table 6). Mass fractionof Cr in seminal fluid of normal prostate was found to be 0.41 mg/kg, inBPH 0.55 mg/kg, and in PCa 1.2 mg/kg on dry mass basis (Table 6). Theupper limit for Cr mass fraction in dry seminal fluid from normal or BPHsubject was determined to be 0.90 mg/kg on dry mass basis.

If PCa needs to be discriminated from normal and BPH and if Cr contentin a seminal fluid sample prepared and analysed as described in theExample 18 exceeds 0.90 mg/kg dry tissue, prostate carcinoma with anaccuracy of 94±3% can be diagnosed.

Example 26 Establishing the Prostate Condition Using Mg Mass Fraction ina Seminal Fluid Sample

The tissue content of Mg was found to be significantly different in mostcancerous seminal fluid samples as compared to normal and benignhyperplastic seminal fluid samples (Example 18, Table 6). Mass fractionof Mg in seminal fluid of normal prostate was found to be 1100 mg/kg, inBPH 3674 mg/kg, and in PCa 140 mg/kg on dry mass basis (Table 6). Thelower limit for Mg mass fraction in seminal fluid from normal or BPHsubject was determined to be 700 mg/kg on dry mass basis.

If PCa needs to be discriminated from normal and BPH and if Mg contentin a seminal fluid sample prepared and analysed as described in theExample 18 does not exceed 700 mg/kg dry tissue, prostate carcinoma withan accuracy of 92±5% can be diagnosed.

Example 26 Establishing the Prostate Condition Using Ca Mass Fraction ina Seminal Fluid Sample

The tissue content of Ca was found to be significantly different in mostcancerous seminal fluid samples as compared to normal and benignhyperplastic seminal fluid samples (Example 18, Table 6). Mass fractionof Ca in seminal fluid of normal prostate was found to be 3030 mg/kg, inBPH 8237 mg/kg, and in PCa 1300 mg/kg on dry mass basis (Table 6). Thelower limit for Ca mass fraction in dry seminal fluid from normal or BPHsubject was determined to 2200 mg/kg on dry mass basis.

If PCa needs to be discriminated from normal and BPH and if Ca contentin a seminal fluid sample prepared and analysed as described in theExample 18 does not exceed 2200 mg/kg dry tissue, prostate carcinomawith an accuracy of 90±5% can be diagnosed.

Example 27 Determination of Mass Fraction Levels of 44 Elements Relativeto the Mass Fraction of Calcium in Normal, Cancerous and BPH EPS.

Mass fraction ratios of the elements mentioned in the Example 17 aredifferent in non-cancerous and cancerous EPS and therefore these can beused as prostate tumor biomarkers. In the Table 7 mass fraction ratiosof 44 elements relative to mass fraction of calcium are presented.Further, ratios of the mass fraction ratios for EPS from normal, BPH andcancerous subjects are given. The data in the Table 7 allow evaluatingthe importance of individual mass fraction ratios of 44 elementsrelative to the mass fraction of calcium for the diagnosis of PCa.

TABLE 7 Means of ratios and their ratios between mean values of massfraction ratios of Ca to mass fractions of other chemical elements inEPS from normal, benign hyperplastic (BPH) and cancerous (PCa) subjects.Ratios of means Prostatic fluid BPH to PCa to PCa to Element Normal BPHPCa Normal Normal BPH Ca/Li 24530 60481 18046 2.5 0.7 0.3 Ca/B 113284233 3800 0.4 0.3 0.9 Ca/Na 0.2 0.2 0.0 0.9 0.1 0.1 Ca/Mg 2.2 2.4 2.01.1 0.9 0.8 Ca/Al 2386 861 87 0.4 0.04 0.1 Ca/Si 428 243 35 0.6 0.1 0.1Ca/P 6.4 2.2 2.1 0.3 0.3 1.0 Ca/S 2.0 2.1 0.6 1.1 0.3 0.3 Ca/K 0.3 0.40.2 1.3 0.7 0.5 Ca/Sc 192704 195808 380000 1.0 2.0 1.9 Ca/Cr 18189 11976160 0.7 0.01 0.01 Ca/Mn 10931 15155 962 1.4 0.09 0.1 Ca/Fe 703 407 100.6 0.01 0.03 Ca/Co 327111 236917 44829 0.7 0.1 0.2 Ca/Ni 34350 2350 5410.1 0.02 0.2 Ca/Cu 1304 788 263 0.6 0.2 0.3 Ca/Zn 1.3 2.1 1.9 1.6 1.50.9 Ca/Se 7072 7429 38000 1.1 5.4 5.1 Ca/Br 347 247 7 0.7 0.02 0.03Ca/Rb 209 333 159 1.6 0.8 0.5 Ca/Sr 4074 4179 1206 1.0 0.3 0.3 Ca/Y1062560 769711 138346 0.7 0.1 0.2 Ca/Zr 160360 75539 8171 0.5 0.1 0.1Ca/Ag 425598 28803 4046 0.1 0.01 0.1 Ca/Sb 110400 21303 38000 0.2 0.31.8 Ca/Cs 141702 126577 36370 0.9 0.3 0.3 Ca/Ba 31319 30715 1148 1.00.04 0.04 Ca/La 199512 549579 84542 2.8 0.4 0.2 Ca/Ce 859311 69970748008 0.8 0.1 0.1 Ca/Pr 200727 1005734 174020 5.0 0.9 0.2 Ca/Nd 991023389312 91545 0.4 0.1 0.2 Ca/Sm 1104000 1019233 156230 0.9 0.1 0.2 Ca/Gd1104000 903780 106736 0.8 0.1 0.1 Ca/Tb 1104000 1075800 263046 1.0 0.20.2 Ca/Dy 1104000 1091721 198095 1.0 0.2 0.2 Ca/Ho 1104000 1075800315152 1.0 0.3 0.3 Ca/Er 1104000 1075800 151557 1.0 0.1 0.1 Ca/Au 15419057363 380000 0.4 2.5 6.6 Ca/Cd 431048 126876 10313 0.3 0.02 0.1 Ca/Hg143862 215160 76000000 1.5 528.3 353.2 Ca/Tl 995043 1075800 24762 1.10.02 0.02 Ca/Pb 130574 33968 3684 0.3 0.03 0.1 Ca/Bi 671533 1056778 94461.6 0.01 0.01 Ca/Th 431082 926283 86428 2.1 0.2 0.1 Ca/U 1104000 107580079838 1.0 0.1 0.1

Example 27 Determination of Mass Fraction Levels of 44 Elements Relativeto the Mass Fraction of Zinc in Normal, Cancerous and BPH EPS.

Mass fraction ratios of the elements mentioned in the Example 17 aredifferent in non-cancerous and cancerous EPS and therefore these can beused as prostate tumor biomarkers. In the Table 8 mass fraction ratiosof 44 elements relative to mass fraction of zinc are presented. Further,ratios of the mass fraction ratios for EPS from normal, BPH andcancerous subjects are given. The data in the Table 8 allow evaluatingthe importance of individual mass fraction ratios of 44 elementsrelative to the mass fraction of zinc for the diagnosis of PCa.

TABLE 8 Means of ratios and their ratios between mean values of massfraction ratios of Zn to mass fractions of other chemical elements inEPS from normal, benign hyperplastic (BPH) and cancerous (PCa) subjects.Mass Ratios of means fraction Prostatic fluid BPH to PCa to PCa to ratioNormal BPH PCa Normal Normal BPH Zn/Li 19121 28666 9498 1.50 0.50 0.33Zn/B 8830 2006 2000 0.23 0.23 1.00 Zn/Na 0.2 0.1 0.0 0.56 0.06 0.11Zn/Mg 1.7 1.1 1.1 0.67 0.63 0.94 Zn/Al 1860.0 407.9 45.6 0.22 0.02 0.11Zn/Si 333.6 115.0 18.2 0.34 0.05 0.16 Zn/P 5.0 1.1 1.1 0.21 0.22 1.06Zn/S 1.6 1.0 0.3 0.64 0.20 0.32 Zn/K 0.2 0.2 0.1 0.82 0.46 0.57 Zn/Ca0.8 0.5 0.5 0.61 0.68 1.11 Zn/Sc 150218 92808 200000 0.62 1.33 2.15Zn/Cr 14179 5676 84 0.40 0.01 0.01 Zn/Mn 8521 7183 506 0.84 0.06 0.07Zn/Fe 548.2 193.0 5.4 0.35 0.01 0.03 Zn/Co 254993 112292 23594 0.44 0.090.21 Zn/Ni 26777 1114 285 0.04 0.01 0.26 Zn/Cu 1017 374 138 0.37 0.140.37 Zn/Se 5513 3521 20000 0.64 3.63 5.68 Zn/Br 270.3 117.2 3.6 0.430.01 0.03 Zn/Rb 162.9 157.7 83.8 0.97 0.51 0.53 Zn/Sr 3175 1981 635 0.620.20 0.32 Zn/Y 828296 364822 72814 0.44 0.09 0.20 Zn/Zr 125005 358034301 0.29 0.03 0.12 Zn/Ag 331766 13652 2129 0.04 0.01 0.16 Zn/Sb 8606010097 20000 0.12 0.23 1.98 Zn/Cs 110461 59994 19142 0.54 0.17 0.32 Zn/Ba24414 14558 604 0.60 0.02 0.04 Zn/La 155525 260485 44496 1.67 0.29 0.17Zn/Ce 669858 331642 25267 0.50 0.04 0.08 Zn/Pr 156473 476691 91590 3.050.59 0.19 Zn/Nd 772531 184524 48182 0.24 0.06 0.26 Zn/Sm 860600 48308982226 0.56 0.10 0.17 Zn/Gd 860600 428367 56177 0.50 0.07 0.13 Zn/Tb860600 509900 138445 0.59 0.16 0.27 Zn/Dy 860600 517446 104261 0.60 0.120.20 Zn/Ho 860600 509900 165869 0.59 0.19 0.33 Zn/Er 860600 509900 797670.59 0.09 0.16 Zn/Au 120196 27189 200000 0.23 1.66 7.36 Zn/Cd 33601460136 5428 0.18 0.02 0.09 Zn/Hg 112145 101980 40000000 0.91 356.68392.23 Zn/Tl 775665 509900 13033 0.66 0.02 0.03 Zn/Pb 101786 16100 19390.16 0.02 0.12 Zn/Bi 523479 500884 4972 0.96 0.01 0.01 Zn/Th 336041439033 45488 1.31 0.14 0.10 Zn/U 860600 509900 42020 0.59 0.05 0.08

Example 27 Determination of Mass Fraction Levels of 44 Elements Relativeto the Mass Fraction of Calcium in Seminal Fluid From Normal, Cancerousand BPH Subjects

Mass fraction ratios of the elements mentioned in the Example 18 aredifferent in non-cancerous and cancerous seminal fluid and thereforethese can be used as prostate tumor biomarkers. In the Table 9 massfraction ratios of 44 elements relative to mass fraction of calcium arepresented. Further, ratios of the mass fraction ratios for seminal fluidfrom normal, BPH and cancerous subjects are given. The data in the Table9 allow evaluating the importance of individual mass fraction ratios of44 elements relative to the mass fraction of calcium for the diagnosisof PCa.

TABLE 9 Means of ratios and their ratios between mean values of massfraction ratios of Ca to mass fractions of other chemical elements inseminal fluid derived from normal, benign hyperplastic (BPH) andcancerous (PCa) subjects. Mass Ratios of means fraction Seminal fluidBPH to PCa to PCa to ratio Normal BPH PCa Normal Normal BPH Ca/Li 8258448711 109809 0.6 1.3 2.3 Ca/B 3801 1967 1300 0.5 0.3 0.7 Ca/Na 0.1 0.20.04 1.4 0.3 0.2 Ca/Mg 2.8 2.2 9.3 0.8 3.4 4.1 Ca/Al 794 1360 277 1.70.3 0.2 Ca/Si 481 281 109 0.6 0.2 0.4 Ca/P 0.3 1.0 0.1 3.3 0.3 0.1 Ca/S1.5 1.7 0.5 1.1 0.3 0.3 Ca/K 0.6 0.4 0.3 0.7 0.6 0.9 Ca/Sc 65758 196333130000 3.0 2.0 0.7 Ca/Cr 7331 15114 1079 2.1 0.1 0.1 Ca/Mn 13462 2327710428 1.7 0.8 0.4 Ca/Fe 160 476 152 3.0 1.0 0.3 Ca/Co 303400 284857130000 0.9 0.4 0.5 Ca/Ni 13565 2520 13050 0.2 1.0 5.2 Ca/Cu 1403 6501316 0.5 0.9 2.0 Ca/Zn 4.2 2.1 9.3 0.5 2.2 4.5 Ca/Se 4480 5858 3567 1.30.8 0.6 Ca/Br 99 194 24 2.0 0.2 0.1 Ca/Rb 431 342 260 0.8 0.6 0.8 Ca/Sr6691 3614 2625 0.5 0.4 0.7 Ca/Y 221025 823700 130000 3.7 0.6 0.2 Ca/Zr39388 109571 28524 2.8 0.7 0.3 Ca/Ag 303400 823700 43458 2.7 0.1 0.1Ca/Sb 30340 82370 130000 2.7 4.3 1.6 Ca/Cs 219853 131731 95502 0.6 0.40.7 Ca/Ba 22721 32499 9663 1.4 0.4 0.3 Ca/La 30340 830622 130000 27.44.3 0.2 Ca/Ce 267497 739297 140752 2.8 0.5 0.2 Ca/Pr 303400 823700130000 2.7 0.4 0.2 Ca/Nd 303400 823700 130000 2.7 0.4 0.2 Ca/Sm 303400823700 830635 2.7 2.7 1.0 Ca/Gd 303400 823700 130000 2.7 0.4 0.2 Ca/Tb303400 823700 130000 2.7 0.4 0.2 Ca/Dy 303400 823700 130000 2.7 0.4 0.2Ca/Ho 303400 823700 130000 2.7 0.4 0.2 Ca/Er 303400 823700 130000 2.70.4 0.2 Ca/Au 134556 116232 130000 0.9 1.0 1.1 Ca/Cd 252833 324398 687681.3 0.3 0.2 Ca/Hg 84303 164740 130000 2.0 1.5 0.8 Ca/Tl 303400 823700130000 2.7 0.4 0.2 Ca/Pb 30681 58599 15384 1.9 0.5 0.3 Ca/Bi 232725823700 78432 3.5 0.3 0.1 Ca/Th 169944 823769 130000 4.8 0.8 0.2 Ca/U303400 823700 130000 2.7 0.4 0.2

Example 28 Determination of Mass Fraction Levels of 44 Elements Relativeto the Mass Fraction of Zinc in Seminal Fluid Derived from Normal,Cancerous and BPH Subjects

Mass fraction ratios of the elements mentioned in the Example 18 aredifferent in non-cancerous and cancerous seminal fluid and thereforethese can be used as prostate tumor biomarkers. In the Table 10 massfraction ratios of 44 elements relative to mass fraction of zinc arepresented. Further, ratios of the mass fraction ratios for normalseminal fluid, BPH and cancerous seminal fluid are given. The data inthe Table 10 allow evaluating the importance of individual mass fractionratios of 44 elements relative to the mass fraction of calcium for thediagnosis of PCa.

TABLE 10 Means of ratios and their ratios between mean values of massfraction ratios of Zn to mass fractions of other chemical elements inseminal fluid derived from normal, benign hyperplastic (BPH) andcancerous (PCa) subjects. Mass Ratios of means fraction Seminal fluidBPH to PCa to PCa to ratio Normal BPH PCa Normal Normal BPH Zn/Li 1989823655 11826 1.19 0.59 0.50 Zn/B 916 955 140 1.04 0.15 0.15 Zn/Na 0.030.1 0.004 2.76 0.12 0.04 Zn/Mg 1.6 1.1 1.0 0.67 0.62 0.92 Zn/Al 191.3660.2 29.8 3.45 0.16 0.05 Zn/Si 115.8 136.3 11.7 1.18 0.10 0.09 Zn/P 0.10.5 0.01 6.70 0.13 0.02 Zn/S 0.4 0.8 0.1 2.28 0.14 0.06 Zn/K 0.1 0.20.04 1.33 0.26 0.20 Zn/Ca 0.4 0.5 0.1 1.10 0.24 0.22 Zn/Sc 15844 9534214000 6.02 0.88 0.15 Zn/Cr 1766 7339 116 4.16 0.07 0.02 Zn/Mn 3243 113031123 3.49 0.35 0.10 Zn/Fe 38.5 231.0 16.4 6.00 0.43 0.07 Zn/Co 73100138331 14000 1.89 0.19 0.10 Zn/Ni 3268 1224 1405 0.37 0.43 1.15 Zn/Cu338 316 142 0.93 0.42 0.45 Zn/Se 1079.4 2844.5 384.1 2.64 0.36 0.14Zn/Br 23.8 94.1 2.6 3.96 0.11 0.03 Zn/Rb 103.9 166.2 28.0 1.60 0.27 0.17Zn/Sr 1612 1755 283 1.09 0.18 0.16 Zn/Y 53253 400000 14000 7.51 0.260.04 Zn/Zr 9490 53209 3072 5.61 0.32 0.06 Zn/Ag 73100 400000 4680 5.470.06 0.01 Zn/Sb 7310 40000 14000 5.47 1.92 0.35 Zn/Cs 52970 63970 102851.21 0.19 0.16 Zn/Ba 5474 15782 1041 2.88 0.19 0.07 Zn/La 7310 40336114000 55.18 1.92 0.03 Zn/Ce 64450 359013 15158 5.57 0.24 0.04 Zn/Pr73100 400000 14000 5.47 0.19 0.04 Zn/Nd 73100 400000 14000 5.47 0.190.04 Zn/Sm 73100 400000 89453 5.47 1.22 0.22 Zn/Gd 73100 400000 140005.47 0.19 0.04 Zn/Tb 73100 400000 14000 5.47 0.19 0.04 Zn/Dy 73100400000 14000 5.47 0.19 0.04 Zn/Ho 73100 400000 14000 5.47 0.19 0.04Zn/Er 73100 400000 14000 5.47 0.19 0.04 Zn/Au 32419 56444 14000 1.740.43 0.25 Zn/Cd 60917 157532 7406 2.59 0.12 0.05 Zn/Hg 20312 80000 140003.94 0.69 0.18 Zn/TI 73100 400000 14000 5.47 0.19 0.04 Zn/Pb 7392 284561657 3.85 0.22 0.06 Zn/Bi 56072 400000 8447 7.13 0.15 0.02 Zn/Th 40946400033 14000 9.77 0.34 0.03 Zn/U 73100 400000 14000 5.47 0.19 0.04

Example 29 Using the Ca/Mn Mass Fraction Ratio in EPS to EstablishProstate Condition

The Ca/Mn mass fraction ratio in EPS was found to be significantlydifferent in most cancerous EPS as compared to normal and benignhyperplastic EPS. The upper limit for Ca/Mn mass fraction ratio on drymass basis in cancerous EPS was determined to be 1900 (Table 7).

If PCa needs to be discriminated from normal and BPH and if the Ca/Mnratio in the EPS sample prepared and analysed as described in Example 17does not exceed 1900, prostate carcinoma with an accuracy exceeding 96%can be diagnosed.

Example 30 Using the Ca/Al Mass Fraction Ratio in Seminal Fluid toEstablish Prostate Condition

The Ca/Al mass fraction ratio in seminal fluid was found to besignificantly different in most cancerous seminal fluid as compared tonormal and benign hyperplastic seminal fluid. The upper limit for Ca/Almass fraction ratio on dry mass basis in cancerous seminal fluid wasdetermined to be 290 (Table 9).

If PCa seminal fluid needs to be discriminated from normal and BPH oneand if the Ca/Al ratio in the seminal fluid sample prepared and analysedas described in Example 18 does not exceed 290, prostate carcinoma withan accuracy exceeding 98% can be diagnosed.

Example 31 Using the Zn/Cu Mass Fraction Ratio in EPS to EstablishProstate Condition

The Zn/Cu mass fraction ratio in EPS was found to be significantlydifferent in most cancerous EPS as compared to normal and benignhyperplastic EPS. The upper limit for Zn/Cu mass fraction ratio on drymass basis in cancerous EPS was determined to be 165 (Table 8).

If PCa EPS needs to be discriminated from normal and BPH EPS and if theZn/Cu ratio in the EPS sample prepared and analysed as described inExample 17 does not exceed 165, prostate carcinoma with an accuracy of95% can be diagnosed.

Example 32 Using the Zn/Cu Mass Fraction Ratio in Seminal Fluid toEstablish Prostate Condition

The Zn/Cu mass fraction ratio in seminal fluid was found to besignificantly different in most cancerous seminal fluids as compared tonormal and benign hyperplastic seminal fluid. The upper limit for Zn/Cumass fraction ratio on dry mass basis in cancerous seminal fluid wasdetermined to be 155 (Table 10).

If PCa EPS needs to be discriminated from normal and BPH EPS and if theZn/Cu ratio in the EPS sample prepared and analysed as described inExample 18 does not exceed 155, prostate carcinoma with an accuracybetter than 95% can be diagnosed.

Example 33 Using the Ca*Mg*Zn/Mn*Bi*Se Mass Fraction Ratio Combinationin EPS to Establish Prostate Condition

The Ca*Mg*Zn/Mn*Bi*Se mass fraction ratio in EPS was found to besignificantly different in most cancerous EPS as compared to normal andbenign hyperplastic EPS. The lower limit for Ca*Mg*Zn/Mn*Bi*Se massfraction ratio on dry mass basis in healthy EPS was determined to be2E8.

If PCa EPS needs to be discriminated from normal and BPH EPS and if theCa*Mg*Zn/Mn*Bi*Se ratio in the EPS sample prepared and analysed asdescribed in Example 18 is below 2E8, prostate carcinoma with anaccuracy better than 95% can be diagnosed.

Example 34 Using the Ca*Mg*Zn/Mn*Bi*Se Mass Fraction Ratio Combinationin Seminal Fluid to Establish Prostate Condition.

The Ca*Mg*Zn/Mn*Bi*Se mass fraction ratio in seminal fluid was found tobe significantly different in most cancerous seminal fluids as comparedto normal and benign hyperplastic seminal fluid. The lower limit forCa*Mg*Zn/Mn*Bi*Se mass fraction ratio on dry mass basis in healthyseminal fluid was determined to be 2E6.

If PCa seminal fluid needs to be discriminated from normal and BPHseminal fluid and if the Ca*Mg*Zn/Mn*Bi*Se ratio in the seminal fluidsample prepared and analysed as described in Example 19 is below 2E6,prostate carcinoma with an accuracy better than 95% can be diagnosed.

Example 35 Using the Ca/Ba Mass Fraction Ratio to Establish ProstateCondition

The Ca/Ba mass fraction ratio was found to be significantly different inmost cancerous prostate tissues as compared to normal and benignhyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Bamass fraction ratio on dry mass basis in cancerous prostate tissue wasdetermined to be M+3SD (M—arithmetic mean, SD—standard deviation) or 400(FIG. 13).

If PCa needs to be discriminated from normal and BPH tissue and if theCa/Ba ratio in a prostate biopsy sample prepared and analysed asdescribed in Example 1 does not exceed 400, prostate carcinoma with anaccuracy of 100-2% can be diagnosed. The sensitivity and specificity ofthe Ca/Ba ratio based test is 100-9% and 100-2%, respectively.

Example 36 Using the Ca/P Mass Fraction Ratio to Establish ProstateCondition

The Ca/P mass fraction ratio was found to be significantly different inmost cancerous prostate tissues as compared to normal and benignhyperplastic tissues (Example 8, Table 2). The upper limit for Ca/P massfraction ratio on dry mass basis in cancerous prostate tissue wasdetermined to be M+3SD (M—arithmetic mean, SD—standard deviation) or0.15 (FIG. 14).

If PCa needs to be discriminated from normal and BPH tissue and if theCa/P ratio in a prostate biopsy sample prepared and analysed asdescribed in Example 1 does not exceed 0.15, prostate carcinoma with anaccuracy of 98±2% can be diagnosed. The sensitivity and specificity ofthe Ca/P ratio based test is 91±9% and 100-3%, respectively.

Example 37 Using the Ca/Si Mass Fraction Ratio to Establish ProstateCondition

The Ca/Si mass fraction ratio was found to be significantly different inmost cancerous prostate tissues as compared to normal and benignhyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Simass fraction ratio on dry mass basis in cancerous prostate tissue wasdetermined to be M+3SD (M—arithmetic mean, SD—standard deviation) or 5(FIG. 15).

If PCa needs to be discriminated from normal and BPH tissue and if theCa/Si ratio in a prostate biopsy sample prepared and analysed asdescribed in Example 1 does not exceed 5, prostate carcinoma with anaccuracy of 98±2% can be diagnosed. The sensitivity and specificity ofthe Ca/Si ratio based test is 91±9% and 100-3%, respectively.

Example 38 Using the Ca/Sr Mass Fraction Ratio to Establish ProstateCondition

The Ca/Sr mass fraction ratio was found to be significantly different inmost cancerous prostate tissues as compared to normal and benignhyperplastic tissues (Example 8, Table 2). The upper limit for Ca/Srmass fraction ratio on dry mass basis in cancerous prostate tissue wasdetermined to be M+3SD (M—arithmetic mean, SD—standard deviation) or 250(FIG. 16).

If PCa needs to be discriminated from normal and BPH tissue and if theCa/Sr ratio in a prostate biopsy sample prepared and analysed asdescribed in Example 1 does not exceed 250, prostate carcinoma with anaccuracy of 98±2% can be diagnosed. The sensitivity and specificity ofthe Ca/Sr ratio based test is 91±9% and 100-3%, respectively.

Example 39 Using the Zn/Mn Mass Fraction Ratio to Establish ProstateCondition

The Zn/Mn mass fraction ratio was found to be significantly different inmost cancerous prostate tissues as compared to normal and benignhyperplastic tissues (Example 8, Table 2). The upper limit for Zn/Mnmass fraction ratio on dry mass basis in cancerous prostate tissue wasdetermined to be M+3SD (M—arithmetic mean, SD—standard deviation) or 170(FIG. 17).

If PCa needs to be discriminated from normal and BPH tissue and if theZn/Mn ratio in a prostate biopsy sample prepared and analysed asdescribed in Example 1 does not exceed 170, prostate carcinoma with anaccuracy of 98±2% can be diagnosed. The sensitivity and specificity ofthe Zn/Mn ratio based test is 91±9% and 100-3%, respectively.

Example 40 Using the [(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 Mass FractionRatio Combination to Establish Prostate Condition

The [(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratio combinationwas found to be significantly different in most cancerous prostatetissues as compared to normal and benign hyperplastic tissues. The upperlimit for [(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratiocombination on dry mass basis in cancerous prostate tissue wasdetermined to be M+60SD (M—arithmetic mean, SD—standard deviation) or100000 (FIG. 18).

If PCa needs to be discriminated from normal and BPH tissue and if the[(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratio combination in aprostate biopsy sample prepared and analysed as described in Example 1does not exceed 100000, prostate carcinoma with an accuracy of 100-2%can be diagnosed. The sensitivity and specificity of the[(Zn*Ca*Mg*Cd)/(Si*Br*Al*Ba)]*1000 mass fraction ratio combination basedtest is 100-10% and 100-3%, respectively.

Example 41 Using the Normalized Mass Fraction Ratio Combinations of Ag,Al, Ba,Bi, Br,Ca, Cd, Ce, Co, Cr, Cs, Cu, Hg, K, Li, Mg, Mn, Na, Ni, P,Pb, Rb, S, Sb, Se, Si, Sr and Zn to Establish Prostate Condition fromthe Prostate Tissue Samples

Mass fraction levels of the elements can be normalized to the referencelevels of same elements. Further, combination of normalized massfraction ratios can be used to diagnose prostate condition. Toillustrate this the normalized mass fraction levels for 27 elements werecalculated as mass fraction of the element divided by the median valueof the mass fraction of the same element in the tissue samples takenfrom normal individuals.

The following combination of normalised mass fraction ratios[(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Li_(n)*Mn_(n)*Ni_(n)*Pb_(n)*Sb_(n)*Si_(n)*Sr_(n))]*10¹⁸ was found to besignificantly different in most cancerous prostate tissues as comparedto normal and benign hyperplastic tissues. The upper limit for[(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Li_(n)*Mn_(n)*Ni_(n)*Pb_(n)*Sb_(n)*Si_(n)*Sr_(n))]*10¹⁸ combination ofnormalised mass fraction ratios on dry mass basis in cancerous prostatetissue was determined to be 100000000000 (FIG. 19).

If PCa needs to be discriminated from normal and BPH tissue and if the[(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Li_(n)*Mn_(n)*Ni_(n)*Pb_(n)*Sb_(n)*Si_(n)*Sr_(n))]*10¹⁸ ratio in aprostate biopsy sample prepared and analysed as described in Example 1does not exceed 100000000000, prostate carcinoma with an accuracy of100-2% can be diagnosed. The sensitivity and specificity of the[(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Li_(n)*Mn_(n)*Ni_(n)*Pb_(n)*Sb_(n)*Si_(n)*Sr_(n))]*10¹⁸ ratio based testis 100-10% and 100-3%, respectively.

Example 42 Using the Normalized Mass Fraction Ratio Combinations of Ag,Al, Au, B, Ba, Bi, Br, Ca, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg,Ho, K, La, Li, Mg, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm,Sr, Th, Tl, U, Y, Zn and Zr to Establish Prostate Condition from theProstate Tissue Samples

Mass fraction levels of the elements can be normalized to the referencelevels of same elements. Further, combination of normalized massfraction ratios can be used to diagnose prostate condition. To improvethe diagnostic value of the normalized mass fraction ratio prostatecancer test the number of elements in the combination can be increased.To illustrate this the normalized mass fraction levels for 45 elementswere calculated as mass fraction of the element taken from the list Ag,Al, Au, B, Ba, Bi, Br, Ca, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg,Ho, K, La, Li, Mg, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm,Sr, Th, Tl, U, Y, Zn and Zr and divided by the median value of the massfraction of the same element in the tissue samples taken from normalindividuals.

The[(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Sc_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Au_(n)*B_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Dy_(n)*Er_(n)*Fe_(n)*Gd_(n)*Ho_(n)*La_(n)*Li_(n)*Mn_(n)*Nd_(n)*Ni_(n)*Pb_(n)*Pr_(n)*Sb_(n)*Si_(n)*Sm_(n)*Sr_(n)*Th_(n)*Ti_(n)*U_(n)*Y_(n)*Zr_(n))]*10³⁴ mass fraction ratio combination was found to besignificantly different in most cancerous prostate tissue samples ascompared to normal and benign hyperplastic tissue samples. Thediagnostic window, i.e. the gap between the lowest normalized massfraction ratio combination from BPH group and the highest normalizedmass fraction ratio combination from the prostate cancer group, hasincreased to five orders of magnitude (FIG. 20). The upper limit for[(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Sc_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Au_(n)*B_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Dy_(n)*Er_(n)*Fe_(n)*Gd_(n)*Ho_(n)*La_(n)*Li_(n)*Mn_(n)*Nd_(n)*Ni_(n)*Pb_(n)*Pr_(n)*Sb_(n)*Si_(n)*Sm_(n)*Sr_(n)*Th_(n)*Ti_(n)*U_(n)*Y_(n)*Zr_(n))]*10³⁴ normalized mass fraction ratio combination on dry massbasis in cancerous prostate tissue was determined to be 10²⁴ (FIG. 20).

If PCa needs to be discriminated from normal and BPH tissue and if the[(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Sc_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Au_(n)*B_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Dy_(n)*Er_(n)*Fe_(n)*Gd_(n)*Ho_(n)*La_(n)*Li_(n)*Mn_(n)*Nd_(n)*Ni_(n)*Pb_(n)*Pr_(n)*Sb_(n)*Si_(n)*Sm_(n)*Sr_(n)*Th_(n)*Ti_(n)*U_(n)*Y_(n)*Zr_(n))]*10³⁴ normalised mass fraction ratio combination in a prostatebiopsy sample prepared and analyzed as described in Example 1 does notexceed 10²⁴, prostate carcinoma with an accuracy of 100-2% can bediagnosed. The sensitivity and specificity of the[(Ca_(n)*Cd_(n)*Co_(n)*Hg_(n)*K_(n)*Mg_(n)*Na_(n)*P_(n)*Rb_(n)*S_(n)*Sc_(n)*Se_(n)*Zn_(n))/(Ag_(n)*Al_(n)*Au_(n)*B_(n)*Ba_(n)*Bi_(n)*Br_(n)*Ce_(n)*Cr_(n)*Cs_(n)*Cu_(n)*Dy_(n)*Er_(n)*Fe_(n)*Gd_(n)*Ho_(n)*La_(n)*Li_(n)*Mn_(n)*Nd_(n)*Ni_(n)*Pb_(n)*Pr_(n)*Sb_(n)*Si_(n)*Sm_(n)*Sr_(n)*Th_(n)*Ti_(n)*U_(n)*Y_(n)*Zr_(n))]*10³⁴ normalized mass fraction ratio combination based test is100-10% and 100-3%, respectively.

Example 43 Identification of Cancer Biomarkes in Expressed ProstaticSecretion Using Inductively Coupled Plasma Mass Spectrometry (ICP-MS)and Inductively Coupled Atomic Emission Spectrometry (ICP-AES).

Equipment:

Inductively coupled plasma mass spectrometry instrument Agilent 7500c.

Specimen:

Expressed Prostatic Secretion samples (EPS) from patients with BenignProstate Hyperplasia (BPH) and low-grade prostate adenocarcinoma (PCa)and EPS samples from healthy volunteers were obtained by transrectalprostate massage. The presence or absence of cancer was confirmed byDigital Rectal Examination (DRE), TransRectal Ultrasound Imaging (TRUSI)and microscopic analysis of tissue morphology in biopsies obtained fromthe same patients, where prescribed by the referring physician.

Reagents:

HNO3 (nitric acid 65% for analysis, max. 0.005 ppm Hg, GR, ISO, Merck),H₂O₂ (hydrogen peroxide pure for analysis, Merck), ICP-MS standardsNCSZC73013 (NCS Certified Reference Material), BCR063R (Community Bureauof Reference of the European Comission) and IRMBD151 (LGC Standards,Weisel, Germany).

Protocol:

0.5 mL of HNO₃ was added to freeze-dried EPS samples and the sampleswere left over night at room temperature. After that 0.25 mL of HNO₃ and0.15 mL of H₂O₂ were added to the samples and placed in water bath at95° C. for 30 min. The heat-treated samples were cooled down to the roomtemperature; the soluble fraction was diluted with deionized water to 15mL and transferred to a plastic measuring bottle. Simultaneously, thesame procedure was performed on a sample containing no EPS fluid, andthe resultant solution was used as a blank sample. All samples wereanalyzed by Inductively Coupled Plasma Mass Spectrometry and InductivelyCoupled Plasma Atomic Emission Spectrometry.

The spectrometer parameters and the main parameters of ICP-MSmeasurements: auxiliary air flow rate—0.9 L/min, nebulizer flow rate—0.9L/min, sample update—0.8 mL/min. The spectrometer parameters for ICP-AESmeasurements: generator output power 1,500 W.

Results:

The content of Al, Cd, Cs, Mn, Ni, Rb, S, Se and Si in EPS was analyzedby ICP-MS. The content of Na, Mg, P, S, K, Ca, Fe, Cu, Zn and Ba in EPSwas analyzed by ICP-AES.

Statistically significant differences in mass fraction levels of 18chemical elements (Table 11) were found in samples derived from lowgrade cancerous, benign hyperplastic and normal EPS.

Differences in mass fraction levels of these elements can be used fordiagnosis and therapeutic purpose. The data in Table 5 allow evaluatingthe importance of the individual chemical element content informationfor the diagnosis of clinical prostate cancer (PCa).

TABLE 11 Comparison of median values of chemical element mass fractions(mg · kg⁻¹, dry mass basis) in normal, benign hyperplastic (BPH) and lowgrade cancerous (PCa) EPS. Normal BPH PCa BPH/Normal PCa/Normal PCa/BPHAl 29.20 13.06 91.21 0.4 3.1 7.0 Ba 1.12 0.42 3.23 0.4 2.9 7.8 Ca 99899729 14000 1.0 1.4 1.4 Cd 0.04 0.04 0.03 1.0 0.9 0.9 Cs 0.10 0.08 0.090.9 0.9 1.1 Cu 6.24 5.27 5.83 0.8 0.9 1.1 Fe 25.68 27.54 24.38 1.1 0.90.9 K 33500 29367 48000 0.9 1.4 1.6 Mg 4644 4549 6500 1.0 1.4 1.4 Mn0.50 0.93 1.58 1.8 3.1 1.7 Na 41286 51010 47000 1.2 1.1 0.9 Ni 0.43 0.830.73 1.9 1.7 0.9 P 3350 3800 4400 1.1 1.3 1.2 Rb 40.75 30.93 47.66 0.81.2 1.5 S 5809 6400 10521 1.1 1.8 1.6 Se 1.24 1.26 1.97 1.0 1.6 1.6 Si84.32 105.4 110.0 1.3 1.3 1.0 Zn 5135 4100 8000 0.8 1.6 2.0

Example 44 Determination of Normalized Mass Fraction Levels of Elementsin Normal, BPH and Low Grade Adenocarcinoma EPS

Mass fraction levels of the elements can be normalized to the referencelevels of same elements. In the Table 12 mass fraction ratios of 18elements relative to reference levels of the same elements arepresented. Reference levels in this example represent mean level valuesderived from the group of 10 EPS samples from verified healthyvolunteers. Anybody skilled in the field can appreciate thatcorresponding reference levels must be determined for different patientpopulations.

TABLE 12 Mean mass fraction levels of elements normalized to thereference levels of the same elements in normal, BPH and low gradeadenocarcinomatous EPS. Example Reference levels, mg/kg dry mass BPH/PCa/ PCa/ basis Normal BPH Pca Normal Normal BPH Al 33.89 0.9 1.1 2.61.1 2.6 2.4 Ba 4.27 1.0 1.1 3.3 1.1 3.3 3.1 Ca 9528 1.1 1.0 1.5 1.0 1.51.5 Cd 0.13 0.9 0.7 0.5 0.7 0.5 0.8 Cs 0.12 0.9 0.7 1.1 0.7 1.1 1.6 Cu7.31 1.0 1.2 1.3 1.2 1.3 1.1 Fe 44.66 0.9 1.4 1.0 1.4 1.0 0.8 K 324021.1 1.0 1.6 1.0 1.6 1.7 Mg 4225 1.1 1.0 1.6 1.0 1.6 1.5 Mn 0.94 1.0 1.81.5 1.8 1.5 0.8 Na 39362 1.0 1.5 1.5 1.5 1.5 1.0 Ni 0.74 0.9 2.6 1.5 2.61.5 0.6 P 3901 0.9 1.1 1.0 1.1 1.0 0.9 Rb 40.15 1.1 0.8 1.4 0.8 1.4 1.8S 6471 1.0 1.1 1.6 1.1 1.6 1.5 Se 1.52 1.0 0.9 1.4 0.9 1.4 1.6 Si 75.410.9 1.6 2.6 1.6 2.6 1.6 Zn 4870 1.0 0.8 1.7 0.8 1.7 2.1

The data in the Table 12 allow evaluating the importance of normalizedmass fraction levels for the diagnosis of PCa. To illustrate thisfurther examples are given.

Example 45 Establishing the Prostate Condition Using the Additive IndexBased on the Normalized Mass Fractions of Ca, K, Mg and Zn in EPS

Further, based on the normalized mass fractions of the elementsdetermined as described in Example 44 the Additive Index (AI) ofprostate condition can be calculated, as exemplified here:

AI=(Ca_(n)+K_(n)+Mg_(n)+Zn_(n))−4

where Ca_(n), K_(n), Mg_(n), Zn_(n) represent normalized values, i.e.mass fractions of Ca, K, Mg and Zn in EPS samples of the subject,divided by the reference levels of the same elements. Additive Index wasfound to be significantly different in most cancerous EPS as compared tonormal and benign hyperplastic EPS (Table 13).

If PCa needs to be discriminated from normal and BPH and if the AdditiveIndex in the EPS sample prepared and analyzed as described in Example 43exceeds the value of 1.0, prostate carcinoma with an accuracy exceeding95% can be diagnosed (FIG. 21).

TABLE 13 Comparison of the Additive Indices between the diagnosticgroups. Normal BPH PCa Mean -3e-007 -0.21 2.0 Std. Deviation 1.2 1.0 1.1Std. Error of Mean 0.39 0.28 0.49 Lower 95% Cl of mean -0.9 -0.8 0.6Upper 95% Cl of mean 0.9 0.4 3.3

Example 46 Establishing the Prostate Condition Using the MultiplicativeIndex Based on the Normalized Mass Fractions of Ca, K, Mg and Zn in EPS

Further, based on the normalized mass fractions of the elementsdetermined as described in Example 44 the Multiplicative Index (MI) ofprostate condition can be calculated, as exemplified here:

MI=(Ca_(n)*K_(n)*Mg_(n)*Zn_(n))/4

where Ca_(n) ,K_(n),Mg_(n),Zn_(n) represent mass fractions of Ca, K, Mgand Zn in EPS of the subject normalised to the reference levels.Multiplicative Index was found to be significantly different in mostcancerous EPS as compared to normal and benign hyperplastic EPS (Table14).

If PCa needs to be discriminated from normal and BPH and if theMultiplicative Index in the EPS sample prepared and analysed asdescribed in Example 43 exceeds the value of 0.7, prostate carcinomawith an accuracy exceeding 99% can be diagnosed (FIG. 22).

TABLE 14 Comparison of the Multiplicative Indices between the diagnosticgroups. Normal BPH PCa Mean 0.4 0.2 1.8 Std. Deviation 0.27 0.16 1.3Std. Error of Mean 0.08 0.04 0.6 Lower 95% Cl of mean 0.2 0.1 0.1 Upper95% Cl of mean 0.5 0.3 3.5

Example 47 Establishing the Prostate Condition Using the MultiplicativeIndex Based on the Normalized Mass Fractions of Ca, K, Mg, Rb, S and Znin EPS

Further, based on the normalized mass fractions of the elementsdetermined as described in Example 44 the 6-element Multiplicative Index(MI/6) of prostate condition can be calculated, as exemplified here:

MI/6=(Ca_(n)*K_(n)*Mg_(n)*Rb_(n)*S_(n)*Zn_(n))/6

where Ca_(n),K_(n),Mg_(n), Rb_(n),S_(n) and Zn_(n) represent massfractions of Ca, K, Mg, Rb, S and Zn in EPS of the subject normalised tothe reference levels. Multiplicative Index was found to be significantlydifferent in most cancerous EPS as compared to normal and benignhyperplastic EPS (Table 15).

If PCa needs to be discriminated from normal and BPH and if theMultiplicative Index in the EPS sample prepared and analysed asdescribed in Example 43 exceeds the value of 0.9, prostate carcinomawith an accuracy exceeding 95% can be diagnosed (FIG. 23).

TABLE 15 Comparison of the Multiplicative Indices between the diagnosticgroups. Normal BPH PCa Mean 0.3 0.2 4.1 Std. Deviation 0.3 0.13 6.4 Std.Error of Mean 0.1 0.04 2.9 Lower 95% Cl of mean 0.13 0.07 -3.9 Upper 95%Cl of mean 0.6 0.2 12

Example 48 Identification of Cancer Biomarkes in Expressed ProstaticSecretion Using Energy Dispersive X-Ray Fluorescence (EDXRF)

Equipment and Method:

EDXRF spectrometer consisted of an annular ¹⁰⁹Cd source with an activityof 2.56 GBq, a 25 mm² Si(Li) detector and portable multichannel analyzercombined with a PC. Its resolution was 270 eV at the 5.9 keV line of55Fe-source. The duration of the Zn measurements together with Br, Fe,Rb, and Sr was 60 min. The intensity of Kα-line of Br, Fe, Rb, Sr, andZn for samples and standards was estimated on the basis of calculatingthe total area of the corresponding photopeak in the spectra. Theelement content was calculated by comparing intensities of Kα-lines forsamples and standards.

Specimen:

Expressed Prostatic Secretion samples (EPS) from patients with BenignProstate Hyperplasia (BPH) and adenocarcinoma (PCa) and EPS samples fromhealthy volunteers were obtained by transrectal prostate massage. Thepresence or absence of cancer was confirmed by Digital RectalExamination (DRE), Ultrasound Imaging (TRUSI) and microscopic analysisof tissue morphology in biopsies obtained from the same patients, whereprescribed by the referring physician.

Sample Preparation:

20 μl of the EPS sample were placed on a backing comprised of a thinfilm of transparent polymeric material (Dacron, Mylar, polyethylene orsimilar, thickness<10 μm). The drop of a sample was freeze-dried on abacking until the constant mass.

Results:

The content of Zn, Br, Fe, Rb, and Sr in EPS obtained from 32 healthyvolunteers, 23 BPH patients and 10 prostate adenocarcinoma patients wasanalyzed by EDXRF.

Differences in mass fraction levels of Zn and Rb were found to bestatistically significant in samples derived from cancerous, benignhyperplastic and normal EPS samples.

Combination of these elements can be used for diagnosis and therapeuticpurpose. The product of mass fraction levels of Rb and Zn divided byten, as expressed by the following formula: (Rb*Zn)/10 was found to bethe most informative marker of prostate cancer. The data in Table 16allow evaluating the importance of the combination of mass fractionlevels of Rb and Zn for the diagnosis of clinical prostate cancer (PCa).

If PCa needs to be discriminated from normal and BPH and if the Productindex (Rb*Zn)/10 in the EPS sample prepared and analysed as described inExample 48 exceeds the value of 350, prostate carcinoma with an accuracyexceeding 98% can be diagnosed (FIG. 24).

TABLE 16 Parameters of the importance (sensitivity, specificity andaccuracy) of the Product index (Rb × Zn)/10 in the samples of expressedprostatic secretion for the diagnosis of PCa (an estimation is made for“PCa” or “Intact and BPH”). Upper limit for PCa Sensitivity, %Specificity, % Accuracy, % <350 100-10 100-2 100-2

1. A method of diagnosing a prostate condition in a subject, comprising:determining, in a sample obtained from a subject, levels of a pluralityof constituents selected from the group consisting of Ca, K, Mg, Zn, Ag,Al, Au, B, Ba, Bi, Br, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Fe, Gd, Hg, Ho,La, Li, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S, Sb, Sc, Se, Si, Sm, Sr, Tb,Th, Tl, U, Y, and Zr; and comparing a combination of the levels of theplurality of constituents in the sample with a combination of controllevels of the same plurality of constituents, in which a differencebetween the combinations is indicative of the prostate condition. 2.-18.(canceled)
 19. A method according to claim 1, wherein the prostatecondition is prostate cancer.
 20. A method according to claim 19,wherein the sample obtained from the subject comprises seminal fluid orexpressed prostatic secretion.
 21. A method according to claim 19,comprising determining levels of least five of the constituents.
 22. Amethod according to claim 19, comprising determining levels of at leastsix of the constituents.
 23. A method of diagnosing a prostate conditionin a subject, comprising: determining, in sample obtained from asubject, a level of at least one constituent selected from the groupconsisting of Ca, K, Mg, Ag, Al, Au, B, Ba, Bi, Br, Cd, Ce, Co, Cr, Cs,Cu, Dy, Er, Fe, Gd, Hg, Ho, La, Li, Mn, Na, Nd, Ni, P, Pb, Pr, Rb, S,Sb, Sc, Se, Si, Sm, Sr, Tb, Th, Tl, U, Y and Zr; and comparing the levelof the at least one constituent in the sample with a control level ofthe same at least one constituent, in which a difference between thelevel of the at least one constituent in the sample and the controllevel of the same at least one constituent is indicative of the prostatecondition, wherein the sample comprises seminal fluid or expressedprostatic secretion.
 24. A method according to claim 23, wherein theprostate condition is prostate cancer.